EPH receptor ligands, and uses related thereto

ABSTRACT

The present invention relates to the discovery of a novel EPH receptor ligand, referred to hereinafter as “Elf-1”, which protein has apparently broad involvement in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, and can be used to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.

BACKGROUND OF THE INVENTION

Pattern formation is the activity by which embryonic cells form orderedspatial arrangements of differentiated tissues. The physical complexityof higher organisms arises during embryogenesis through the interplay ofcell-intrinisic lineage and cell-extrinsic signaling. Inductiveinteractions are essential to embryonic patterning in vertebratedevelopment from the earliest establishment of the body plan, to thepatterning of the organ systems, to the generation of diversive celltypes during tissue differentiation (Davidson, E., (1990) Development108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. etal., (1992) Cell 68: 257-270). The effects of developmental cellinteractions are varied. Typically, responding cells are diverted fromone route of cell differentiation to another by inducing cells thatdiffer from both the uninduced and induced states of the respondingcells (inductions). Sometimes cells induce their neighbors todifferentiate like themselves (homoiogenetic induction); in other casesa cell inhibits its neighbors from differentiating like itself. Cellinteractions in early development may be sequential, such that aninitial induction between two cell types leads to a progressiveamplification of diversity. Moreover, inductive interactions occur notonly in embryos, but in adult cells as well, and can act to establishand maintain morphogenetic patterns as well as induce differentiation(J. B. Gurdon (1992) Cell 68:185-199).

Many types of communication take place among animal cells duringembryogenesis, as well as in the maintenance of tissue in adult animals.These vary from long-range effects, such as those of rather stablehormones circulating in the blood and acting on any cells in the bodythat possess the appropriate receptors, however distant they are, to thefleeting effects of very unstable neurotransmitters operating overdistances of only a few microns. Of particular importance in developmentis the class of cell interactions referred to above as embryonicinduction; this includes influences operating between adjacent cells orin some cases over greater than 10 cell diameters (Saxen et al. (1989)Int J Dev Biol 33:21-48; and Gurdon et al. (1987) Development99:285-306). Embryonic induction is defined as in interaction betweenone (inducing) and another (responding) tissue or cell, as a result ofwhich the responding cells undergo a change in the direction ofdifferentiation. This interaction is often considered one of the mostimportant mechanismn in vertebrate development leading to differencesbetween cells and to the organization of cells into tissues and organs.

Receptor tyrosine kinases are apparently involved in many differentprocess including cellular differentiation, proliferation, embryonicdevelopment and, in some cases, neoplastic growth. High affinity bindingof specfic soluble or matrix-associated growth factor ligands can causethe activated receptor to associate with a specific repertoire ofcytoplasmic singnalling molecules that can lead to a cascade ofintracellular signalling resulting in. for example, activation orinactivation of cellular gene programs involved in differentiationand/or growth. Accordingly, peptide growth factors that are ligands forsuch receptor tyrosine kinases are excellent candidates forintercellular signaling molecules with important developmental roles.Indeed, these ligands are known to have potent effects on a wide varietyof cell activities in vitro. including survival. proliferation,differentiation, adhesion, migration and axon guidance. The powerfulsignaling effects of these molecules are further emphasized by theability of both the ligands and the receptors, when activated bymutation or overexpression, to become potent oncogenes and cause drasticcellular transformation (reviewed by Cantley et al. (1991) Cell64:281-302; Schlessinger and Ullrich (1992) Neuron 9:383-391; and Fantlet al. (1993) Annu Rev Biochem 62:453-481).

To illustrate, specific developmental roles have been demonstrated forsome girowth factors or their tyrosine kinase receptors. For example,the c-kit receptor tyrosinie kinase, encoded at the mouse W locus(Chabot et al. (1988) Nature 335:88-89; and Geissler et al. (1988) Cell55:185-192) and its ligand KL, encoded at the mouse Sl locus (Flanaganand Leder (1990) Cell 63:185-194; Copeland et al. (1990) Cell63:175-183; Huang et al. (1990) Cell 63:225-233; and Zsebo et al. (1990)Cell 63:213-224), determine the proliferation, survival, and/ormigration of primordial germ cells, hematopoietic stem cells, and neuralcrest progenitor cells. Other examples are the trk family ligands andreceptors, with highly specific functions in the developing mammaliannervous system (Klein et al. (1993) Cell 75:113-122; and Jones et al.(1994) Cell 76:989-999) and the FGF receptor, implicated in Xenopusmesoderm induction (Amaya et al. (1991) Cell 66:257-270). Ininvertebrates, too, receptor tyrosine kinases and ligands such assevenless, boss, torso, breathless and let-23 are known to play keyroles in processes that range from setting up the primary embryonic axesto specifying the fate of a single cell in the ommatidium (Greenwald andRubin (1992) Cell 68:271-281; Shilo (1992) Faseb J 6:2915-2922; andZipursky et al. (1992) Cold Spring Horbor Synip Qtuani Biol 57:381-389).Taken together, the emerging picture of the developmental functions ofreceptor tyrosine kinases and their ligands is striking in that thesemolecules play key roles at all stages of embryonic development and in aremarkable range of different types of patterning process.

The receptor tyrosine kinases can be divided into families based onstructural homology and. in at least some cases, obvious sharedfunctional characteristics (Fantl et al. (1993) Annu Rev Biochem62:453-481). The family with by far the largest number of known membersis the EPH family. Since the description of the prototype, the EPHreceptor (Hirai et al. (1987) Science 238:1717-1720), sequences havebeen reported for at least ten members of this family, not countingapparently ortholooous receptors found in more than one species.Additional partial sequences, and the rate at which new members arestill being reported, suggest the family is even larger (Maisonpierre etal. (1993) Oncogene 8:3277-3288; Andres et al. (1 994) Oncogene9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al.(1994) Mech Dev 46:87-100; Xu et al. (1994) Development 120:287-299;Zhou et al. (1994) J. Neurosci Res 37:129-143; and references in Tuziand Gullick (1994) Br J Cancer 69:417-421). Remarkably, despite thelarge number of members in the EPH family, all of these molecules wereidentified as orphan receptors without known ligands.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a novel EPH receptorligand, referred to hereinafter as “Elf-1”, which protein has apparentlybroad involvement in the formation and maintenance of ordered spatialarrangements of differentiated tissues in vertebrates, and can be usedto generate and/or maintain an array of different vertebrate tissue bothin vitro and in vivo.

In general, the invention features an Elf-1 polypeptide, preferably asubstantially pure preparation of an Elf-1 polypeptide, or a recombinantElf-1 polypeptide. In preferred embodiments the polypeptide has abiological activity associated with its binding to an EPH receptor,e.g., it retains the ability to bind to a hek-rclated or sek-relatedreceptor, though it may be able to either agnoize or antagonize signaltransduction by the EPH receptor. The polypeptide can be identical tothe polypeptide shown in SEQ ID No: 2, or it can merely be homologous tothat sequence. For instance, the polypeptide preferably has an aminoacid sequence at least 60% homologous to the amino acid sequence in SEQID No: 2, though higher sequence homologies of, for example, 80%, 90% or95% are also contemplated. The polypeptide can comprise the full lengthprotein represented in SEQ ID No: 2, or it can comprise a fragment ofthat protein, which fragment may be, for instance, at least 5, 10, 20,50 or 100 amino acids in length. A preferred Elf-1 polypeptide comprisesCys-69 through Cys-159, or a sequence homologous thereto.

The polypeptide can be glycosylated, or, by virtue of the expressionsystem in which it is produced, or by modification of the proteinsequence to preclude glycosylation, reduced carbohydrate analogs can beprovided. likewise, Elf-1 polypeptides can be venerated which lack anendogenous signal sequence (though this is typically cleaved off even ifpresent in the pro-form of the protein), or which lack aphosphatidylinositol linkage site to preclude addition ofphosphatidylinositol. In the instance of the latter, the removal of the(C-terminus may result in a soluble form of the protein. In particular,polypeptides which lack at least the last 15 amino acid residues(truncated at Leu-195) are preferred, though polypeptides which aretruncated anywhere between Thr-182 to Leu-195 are also contemplated.

Moreover, as described below, the polypeptide can be either an agonist(e.g. mimics), or alternatively an antagonist of a biological activityof a naturally occuring form of the protein, e.g., the polypeptide isable to modulate growth and/or differentiation of a cell which expressesan EPH receptor.

In a preferred embodiment, a peptide having at least one biologicalactivity of the subject polypepide may differ in amino acid sequencefrom the sequence in SEQ ID No: 2, but such differences result in amodified protein which functions in the same or similar manner as anative Elf-1 protein or which has the same or similar characteristics ofa native Elf-1 protein. However, homologs of the naturally occuringprotein are contemplated which are antagonistic of the normalphysiological role of the naturally occurring protein. For example, thehomolog may be capable of interfering with the ability ofnaturally-occurring forms of Elf-1 to modulate gene expression, e.g. ofdevelopmentally or growth regulated genes.

In yet other preferred embodiments, the Elf-1 protein is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to Elf-1,e.g. the second polypeptide portion is glutathione-S-transferase, e.g.the second polypeptide portion is an enzymatic activity such as alkalinephosphatase, and is a reagent for detecting Elf-1 receptors.

Yet another aspect of the present invention concerns an immunogencomprising a Elf-1 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for anElf-1 polypeptide; e.g. a humoral response, e.g. an antibody response;e.g. a cellular response. In preferred embodiments, the immunogencomprising an antigenic determinant, e.g. a unique determinant, from aprotein represented by SEQ ID No. 2.

A still further aspect of the present invention features an antibodypreparation specifically reactive with an epitope of the Elf-1immunogen.

Another aspect of the present invention provides a substantiallyisolated nlucleic acid having a nucleotide sequence which encodes anElf-1 polypeptide. In preferred embodiments: the encoded polypeptidespecifically binds an EPH receptor protein and/or is able to eitheragnoize or antagonize signal transduction events mediated by the EPHreceptor. The coding sequence of the nucleic acid can comprise asequence which can be identical to the cDNA shown in SEQ ID No: 1, or itcan merely be homologous to that sequence. For instance, the Elf-1encoding sequence preferably has a sequence at least 60% homologous tothe nucleotide sequence in SEQ ID No: 1, though higher sequencehomologies of, or example, 80%, 90% or 95% are also contemplated. Thepolypeptide encoded by the nucleic acid can comprise the amino acidsequence represented in SEQ ID No: 2 which is the full length protein,or it can comprise a fragment of that nucleic acid, which fragment maybe, for instance, encode a fragment of which is, for example, at least5, 10, 20, 50 or 100 amino acids in length. The polypeptide encoded bythe nucleic acid can be either an agonist (e.g. mimics), oralternatively, an antagonist of a biological activity of a naturallyoccuring form of the protein.

Furthermore, in certain preferred embodiments, the subject Elf-1 nucleicacid will include a transcriptional regulatory sequence, e.g. at leastone of a transcriptional promoter or transcriptional enhancer sequence,which regulatory sequence is operably linked to the Elf-1 gene sequence.Such regulatory sequences can be used in to render the Elf-1 genesequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides of SEQ ID No: 1; preferably to at least 20consecutive nucleotides of SEQ ID No: 1; more preferably to at least 40consecutive nucleotides of SEQ ID No: 1.

The invention also features transgcnic non-human animals. e.(g. mice,rats, rabbits. chickens, frogs or pigs. having a transgene, e.g.,animals which include (and preferably express) a heterologous form of anElf-1 gene described herein, or which misexpress an endogenous Elf-1gene, e.g., an animal in which expression of the subject Elf-1 proteinis disrupted. Such a transgenic animal can serve as an animal model forstudying cellular and tissue disorders comprising mutated ormis-expressed Elf-1 alleles or for use in drug screening.

The invention also provides a probe/primer comprising a substantiallypurified oligontieleotide, wherein the oligonucleotide comprises aregion of nucleotide sequence which hybridizes under stringentconditions to at least 10 consecutive nucleotides of sense or antisensesequence of SEQ ID No: 1, or naturally occurring mutants thereof. Inpreferred embodiments, the probe/primer further includes a label groupattached thereto and able to be detected. The label croup can beselected, e.g., from a group consisting of radioisotopes, fluorescentcompounds, enzymes, and enzyme co-factors. Probes of the invelntioln canbe used as a part of a diagnostic test kit for identifying transformedcells, such as for detecting in a sample of cells isolated from apatient, a level of a nucleic acid encoding the subject Elf-1 proteins;e.g. measuring the Elf-1 mRNA level in a cell, or determining whetherthe gellomic Elf-1 gene has been mutated or deleted. Preferably, theoligonucleotide is at least 10 nucleotides in length, though primers of20, 30, 50, 100, or 150 nucleotides in length are also contemplated.

In yet another aspect, the invention provides an assay for screeningtest compounds for inhibitors, or alternatively, potentiators, of aninteraction between Elf-1 and an EPH receptor. An exemplary methodincludes the steps of (i) combining an EPH receptor, an Elf-1polypeptide, and a test compound, e.g., under conditions wherein, butfor the test compound, the Elf-1 protein and the EPH receptor are ableto interact; and (ii) detecting the formation of a complex whichincludes the Elf-1 protein and the receptor. A statistically significantchange, such as a decrease, in the formation of the complex in thepresence of a test compound (relative to what is seen in the absence ofthe test compound) is indicative of a modulation, e.g., inhibition, ofthe interaction between Elf-1 and the receptor. For example, primaryscreens are provided in which the Elf-1 protein and the receptor proteinare combined in a cell-free system and contacted with the test compound;i.e. the cell-free system is selected from a group consisting of a celllysate and a reconstituted protein mixture.

Another aspect of the present invention relates to a method of induicinland/or maintaining a differentiated state, causing proliferation, and/orenhancing survival of a cell responsive to a Elf-1 protein, bycontacting the cells with an Elf-1 agonist or an Elf-1 antagonist. Forexample, the present method is applicable to cell culture technique,such as in the culturing of neuronal and other cells whose survival ordifferentiative state is dependent on Elf-1 fuiction. Moreover, Elf-1agonists and antagonists can be used for therapeutic intervention, suchas to enhance survival and maintenance of neurons and other neural cellsin both the central nervous system and the peripheral nervous system. aswell as to influence other vertebrate organogenic pathways, such asother ectodermal patterning, as well as certain mesodermal andendodermal differentiation processes.

Another aspect of the present invention provides a method of determiningif a subject, e.g. a human patient, is at risk for a disordercharacterized by unwanted cell proliferation or abherent control ofdifferentiation. The method includes detecting, in a tissue of thesubject, the presence or absence of a genetic lesion characterized by atleast one of (i) a mutation of a gene encoding an Elf-1 protein, e.g.represented in SEQ ID No: 2, or a homolog, thereof; or (ii) themis-expression of an Elf-1 gene, In preferred embodiments, detecting thegenetic lesion includes ascertaining the existence of at least one of: adeletion of one or more nucleotides from an Elf-1 gene; an addition ofone or more nucleotides to the gene, a substitution of one or morenucleotides of the gene, a gross chromosomal rearrangement of the gene;an alteration in the level of a messenger RNA transcript of the gene;the presence of a non-wild type splicing pattern of a messenger RNAtranscript of the gene; or a non-wild type level of the protein.

For example, detecting the genetic lesion can include (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequence ofan Elf-1 gene, e.g. the nucleic acid represented in SEQ ID No: 1, ornaturally occurring mutants thereof or 5′ or 3′ flanking sequencesnaturally associated with the Elf-1 gene; (ii) exposing the probe/primerto nucleic acid of the tissue; and (iii) detecting, by hybridization ofthe probe/primer to the nucleic acid. the presence or absence of thegenetic lesion; e.g. wherein detecting the lesion comprises utilizingthe probe/primer to determine the nucleotide sequence of the Elf-1 geneand, optionally, of the flanking nucleic acid sequences. For instance,the probe/primer can be employed in a polymerase chain reaction (PCR) orin a ligation chain reaction (LCR). In alternate embodiments, the levelof Elf-1 protein is detected in an immunoassay using an antibody whichis specifically immunoreactive with an Elf-1 protein.

Yet another aspect of the invention relates to a novel in silu assay fordetecting receptors or their ligands in tissue samples and whileorganisnis. In general, the “RAP-in Situ” assay (for Receptor AffinityProbe) of the present invention comprises (i) providing a hybridmolecule (the affinity probe) including a receptor, or a receptorligand, covalently bonded to an enzymatically active tag, preferably forwhich chromogenic substrates exist, (ii) contacting the tissue ororganism with the affinity probe to form complexes between the probe anda cognate receptor or ligand in the sample, removing unbound probe, and(iii) detecting the affinity complex using a chromogenic substrate forthe enzymatic acitivity associated with the affinity probe. In preferredembodiments, an alkaline phosphatase provides an enzymatic tag, thoughsuch enzymes horseradish peroxidase, β-galactosidase. malatedehydrogenase, yeast alcohol dehydrogenase, α-glycerophosphatedehydrogenase. triose phosphate isomerase, asparaginase, glucoseoxidase, and urcase arc also useful. The method can be used, forexample, to detect patterns of expression for particular receptors andtheir ligands. for measuring the affinity of receptor/ligandinteractions in tissue samples, as well as for generating drug screeningassays in tissue samples. Moreover, the affinity probe can also be usedin diagnostic screening to determine whether a receptor, e.g. an EPHreceptor, or its ligand, e.g. Elf-1 or B61 or LERK-2 protein, aremisexpressed.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195: Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslaition (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AninialCells (R. I. Freshney. Alan R. Liss, Inc., 1987); Immobilized Cell AndEnzymes (IRL, Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzynmoloy (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1A graphically illustrates the Mek4-AP and Sek-AP soluble receptoraffinity reagents. The Mek4 and Sek receptor tyrosine kinases areillustrated on the left. Like other members of the Eph family, each hasa cysteine-rich domain with a characteristic spacing of cysteineresidues (hatched box) followed by two fibronectin III motifs (openboxes) a single transmembrane domain and an intracellular tyrosinekinase domain (filled box). The diagram to the right illustrates thestructure of the Mek4-AP and Sek-AP soluble receptor affinity reagents,each of which consists of the receptor extracellular domain fused to aplacental alkaline phosphatase tag.

FIG. 1B demostrates that the Mek4-AP and Sek-AP fusion proteins areexpressed and secreted into the supernatants of transfected NIH-3T3cells. Cells were metabolically labeled with ³⁵S-methionine, then thesupernatants were immunoprecipitated with a monoclonal antibody againsthuman placental alkaline phosphatase, separated on a 10% polyacrylamidegel, and autoradiographed. Unfused AP is shown for comparison.

FIG. 2A is the nucleic acid sequence of an Elf-1 cDNA clone (SEQ ID No.1), and the virtual amino acid sequence (SEQ ID No. 2). The deducedamino acid sequence is shown in single-letter code above the nucleotidesequence. Cysteine residues are marked with asterisks, potentialN-linked glycosylation sites are indicated by small filled circles, anda probable endpoint for the secretion signal sequence is indicated by anopen triangle. A polyadenylation signal in the 3′ untranslated sequenceis underlined.

FIG. 2B is a hydrophobicity plot of the predicted Elf-1 polypeptide.

FIGS. 3A-F illustrate quantitative cell surface binding of mek4-AP andsek-AP. Cells were treated with supernatants containing mek4-AP, sek-APor unfused AP as a control. The cells were then washed, lysed andassayed colorimetrically for bound AP activity. (FIGS. 3A and 3D)) Cellswere treated with saturating amounts of mek4-AP or sek-AP, or with AP,each at 500 OD/hr/ml. Columns show the average of two bindingdeterminations, and error bars indicate the difference between the two.(FIGS. 3B-C and 3E-F) show the Scatchard analyses of binding. (FIGS.3A-3C illustrate binding to COS cells transfected with Elf-1, and FIGS.3D-F show endogenous ligand expression by the BRL-3A cell line). Bindingcharacteristics calculated for the experiments shown are as follows: formek4-AP cells, 1.0×10⁵ sites average per cell with K_(D)=1.8×10⁻⁸ M;with BRL-3A cells, 3×10⁴ sites per cell with K_(D)=0.67×10⁻⁸ M.

DETAILED DESCRIPTION OF THE INVENTION

Growth factors that are ligands for receptor tyrosine kinases control awide variety of cellular activities. Virtually all of these ligands thathave been characterized are known to have important functions indevelopment and/or physiology and, in at least some cases, to be usefulclinically. The existence of many additional, hitherto unidentifiedligands is implied by the discovery over the last few years of a largenumber of tyrosine kinases that appear by their structure to be cellsurface receptors, yet have no known ligand. The rapid discovery ofthese orphan receptors has been possible mostly through the applicationof techniques such as polymerase chain reaction that take advantage ofthe strong sequence conservation of the kinase catalytic domain.However, in contrast, identification of the ligands for the orphanreceptor tyrosine kinases has been more problematic.

It is also generally accepted that intercellular signaling plays a keyrole throughout vertebrate development. A great deal of progress hasbeen made in understanding signals that mediate some of the earliestpatterning events. However, little is known about the signals thatregulate many of the important events that unfold as gastrulation andearly organogenesis proceed, particularly the cell-cell signalingmolecules that control the expression of gene programs. Protein tyrosinekinases, such as members of the EPH family, have been especiallyintriguing in this regard, particularly because the expression domainsfor several of these receptors include these stages of development.

The expression patterns determined for some of the EPH family receptorshave implied important roles for these molecules in early vertebratedevelopment. In particular, the timing and pattern of expression of sek,mek4 and some of the other receptors during the phase of gastrulationand early organogenesis has suggested functions for these receptors inthe important cellular interactions involved in patterning the embryo atthis stage (Gilardi-Hebenstreit et al. (1992) Oncogene 7:2499-2506;Nieto et al. (1992) Development 16:1137-1150; Henkemeyer et al., supra;Ruiz et al., supra; and Xu et al., supra). Sek, for example, shows anotable early expression in the two areas of the mouse embryo that showobvious segmentation, namely the somites in the mesoderm and therhombomeres of the hindbrain: hence the name sek, for segmentallyexpressed kinase (Gilardi-Hebenstreit et al., supra; Nieto et al.,supra). As in Drosophila, these segmental structures of the mammalianembryo are implicated as important elements in establishing the bodyplan. The observation that Sek expression precedes the appearance ofmorphological segmentation suggests a role for sek in forming thesesegmental structures, or in determining segment-specific cell propertiessuch as lineage compartmentation (Nieto et al., supra). Moreover, EPHreceptors have been implicated, by their pattern of expression, in thedevelopment and maintenance of nearly every tissue in the embryonic andadult body. For instance, EPH receptors have been detected throughoutthe nervous system, the testes, the cartilaginous model of the skeleton,tooth primordia, the infundibular component of the pituitary, variousepithelia tissues, lung, pancreas, liver and kidney tissues.Observations such as this have been indicative of important and uniqueroles for EPH family kinases in development and physiology, but furtherprogress in understanding their action has been severely limited by thelack of information on their ligands.

However, as described in the appended examples, we have utilized solubleMek4-AP (AP=alkaline phosphatase) and Sek-AP fusion proteins in a uniquescreening assay to clone a novel EPH receptor ligand termed herein“Elf-1”. In addition to identifying this novel ligand, we havecharacterized the spatial distribution of expression of the protein andfind that it is likely to be of central importance in development. Giventhe apparent role of the Elf-1 protein in mediating inductive signalsbetween tissues, the present data suggests that this protein is animportant therapeutic target for modulating growth and developmentalgene programs. For example, binding of an Elf-1 polypeptide of thepresent invention with an EPH receptor can be important for initiatingand establishing diverse programs of differentiation; as well as forproviding a mechanism to ensure developmentally coordinated tissuepatterning. Moreover, it is suggested that certain EPH receptors, e.g.the hek receptor, may also play a role in tumorogenesis. Consequently,the interaction of an EPH receptor with the subject Elf-1 polypeptidesmay be significant in the modulation of cellular homeostasis. in thecontrol of organogenesis, or in the maintenance of differentiatedtissues, as well as in the development of lymphocytic leukemias andother neoplastic disorders.

Accordingly, certain aspects of the present invention relate todiagnostic and therapeutic assays and reagents for detecting andtreating disorders involving abhcrent expression of Elf-1. Moreover,drug discovery assays are provided for identifying agents which canmodulate the binding of Elf-1 with EPH receptors. Such agents can beuseful therapeutically to alter the growth and/or differentiation of acell. Other aspects of the invention are described below or will beapparent to those skilled in the art in light of the present disclosure.

For convience, certain terms employed in the specfication, examples, andappended claims are collected here.

As used herein, the terms “EPH receptor” or “EPH-type receptor” refer toa class of receptor tyrosine kinases, comprising at least elevenhomologous genes, though many more orthologs exist within this class,e.g. homologs from different species. EPH receptors are, in general,characterized by an extracellular domain containing a cysteine richregion near the N-terminus and two fibronectin type III repeats (Hiraiet al. (1987) Science 238:1717-1720; Lindberg et al. (1990) Mol CellBiol 10:6316-6324; Chan et al. (1991) Oncogene 6:1057-1061; Maisonpierreet al. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al.(1994) Mech Dev 46:87-100; Xu et al. (1994) Development 120:287-299;Zhou et al. (1994) J. Neurosci Res 37:129-143; and references in Tuziand Gullick (1994) Br Cancer 69:417-421). Exemplary EPH receptorsinclude the eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyrol, tyro4,tyro5, tyro6, tyrol1, cek4, cek5, cek6, cek7, cek8, cek9, cek10, bsk,rtk1, rtk2, rtk3, myk1, myk2, ehk1, ehk2, pagliaccio. htk, erk and nukreceptors. The term “EPH receptor” refers to the membrane form of thereceptor protein, as well as soluble extracellular fragments whichretain the ability to bind the ligand of the present invention.Furthermore, “hek-related receptors” refers to the orthologs of thehuman EPH receptor “hek”, such as cek4, tyro4 and mek4, and may alsoinclude other phylogentically related homologs, such as the eek, bsk,ehk1, ehk2 and cek7 receptors and the family of sek-related receptors.sek. cek(8, pagliaccio, tyro1, and rtk1.

As used herein the term “nucleic acid” refers to polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

As used hereing the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding an Elf-1polypeptide of the present invention, including both exon and(optionally) intron sequences. A “recombinant gene” refers to nucleicacid encoding an Elf-1 polypeptide and comprising Elf-1 encoding exonsequences, though it may optionally include intron sequences which areeither derived from a chlroniosomal Elf-1 gene or from an unrelatedchromosomal gene. An exemplary recombinant gene encoding the subjectElf-1 polypeptide is represented by SEQ ID No: 1. The term “intron”refers to a DNA sequence present in a gliven Elf-1 gene which is nottranslated into protein and is generally found between exons.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of an Elf-1 polypeptideor, where anti-sense expression occurs from the transferred gene, theexpression of a naturally-occurring form of the Elf-1 protein isdisrupted.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome. In the present specification, “plasmid”and “vector” are used interchangeably as the plasmid is the mostcommonly used form of vector. However, the invention is intended toinclude such other forms of expression vectors which serve equivalentfunctions and which become known in the art subsequently hereto.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of a recombinant Elf-1 gene isunder the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring form of the Elf-1 protein.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which effects expressionof the selected DNA sequence in specific cells of a tissue, such ascells of neural origin, e.g. neuronal cells. The term also coversso-called “leaky” promoters, which regulate expression of a selected DNAprimarily in one tissue, but cause expression in other tissues as well.

As used herein a “transgenic animal” is any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous Inucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of the subject Elf-1 protein, e.g. either agonistic or antagonisticforms. However, transgenic animals in which the recombinant Elf-1 geneis silent are also contemplated, as for example, the FLP or CRErecombinase dependent constructs described below. The “non-humananimals” of the invention include vertebrates such as rodents, non-humanprimates, sheep, dog, cow, chickens, amphibians, reptiles, etc.Preferred non-human animals are selected from the rodent familyincluding rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding, forexample, embryogenesis. The term “chimeric animal” is used herein torefer to animals in which the recombinant gene is found, or in which therecombinant is expressed in some but not all cells of the animal. Theterm “tissue-specific chimeric animal” indicates that the recombinantElf-1 gene is present and/or expressed in some tissues but not others.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., an Elf-1 polypeptide), which is partly or entirelyheterologous, i.e., foreign, to the transgenic animal or cell into whichit is introduced, or, is homologous to an endogenous gene of thetransgenic animal or cell into which it is introduced, but which isdesigned to be inserted, or is inserted, into the animal's genome insuch a way as to alter the genome of the cell into which it is inserted(e.g., it is inserted at a location which differs from that of thenatural gene or its insertion results in a knockout). A transgene caninclude one or more transcriptional regulatory sequences and any othernucleic acid, such as introns, that may be necessary for optimalexpression of a selected nucleic acid.

As is well known, genes for a particular polypeptide may exist in singleor multiple copies within the genome of an individual. Such duplicategenes may be identical or may have certain modifications, includingnucleotide substitutions, additions or deletions, which all still codefor polypeptides having substantially the same activity. The term “DNAsequence encoding an Elf-1 polypeptide” may thus refer to one or moregenes within a particular individual. Moreover, certain differences innucleotide sequences may exist between individual organisms, which arecalled alleles. Such allelic differences may or may not result indifferences in amino acid sequence of the encoded polypeptide yet stillencode a protein with the same biological activity.

“Homology” refers to sequence similarity between two peptides or betweentwo nucleic acid molecules. Homology can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences. An“unrelated” or “non-homologous” sequence shares less than 10 percentidentity, though preferably less than percent identity, with an Elf-1sequence of the present invention.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding the subject Elf-1 polypeptide with a second aminoacid sequence defining a domain foreign to and not substantiallyhomologous with any domain of the Elf-1 protein. A chimeric protein maypresent a foreign domain which is found (albeit in a different protein)in an organism which also expresses the first protein, or it may be an“interspecies”, “intergenic”, etc. fusion of protein structuresexpressed by different kinds of organisms. In general, a fusion proteincan be represented by the general formula X-elf-Y, wherein elfrepresents a portion of the protein which is derived from an Elf-1protein, and X and Y are independently absent or represent amino acidsequences which are not related to an Elf-1 sequence.

The term “evolutionarily related to”, with respect to nucleic acidsequences encoding an Elf-1 polypeptide, refers to nucleic acidsequences which have arisen naturally in an organism, includingnaturally occurring mutants. The term also refers to nucleic acidsequences which, while derived from a naturally occurring Elf-1 gene,have been altered by mutagenesis, as for example, the combinatorialmutagenic technigques described below, yet still encode polypeptideswhich have at least one activity of an Elf-1 polypeptide.

The term “isolated” as also used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding thesubject Elf-1 polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks theElf-1 gene in genomic DNA, more preferably no more than 5 kb of suchnaturally occurring flanking sequences, and most preferably less than1.5 kb of such naturally occurring flanking sequence. The term isolatedas used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state.

As described below, one aspect of the invention pertains to an isolatednucleic acid comprising the nucleotide sequence encoding an Elf-1polypeptide, and/or equivalents of such nucleic acids. The term nucleicacid as used herein is intended to include fragments as equivalents. Theterm equivalent is understood to include nucleotide sequences encodingfunctionally equivalent Elf-1 polypeptides or functionally equivalentpeptides which, for example, retain the ability to bind to an tyrosinekinase receptor of the EPH family, e.g. to the mek4-related and/orsek-related receptors. Equivalent nucleotide sequences will includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants; and will, therefore, includesequences that differ from the nucleotide sequence of the Elf-1polypeptide shown in SEQ ID No: 1 due to the degeneracy of the geneticcode. Equivalents will also include nucleotide sequences that hybridizeunder stringent conditions (i.e., equivalent to about 20-27° C. belowthe melting temperature (T_(m)) of the DNA duplex formed in about 1Msalt) to the nuclcotide sequence represented in SEQ ID No: 1. In oneembodiment, equivalents will further include nucleic acid sequencesderived from and evolutionarily related to, a nucleotide sequences shownin any of SEQ ID No: 1.

Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide, homologs of thesubject Elf-1 polypeptide which function in a limited capacity as one ofeither an Elf-1 agonist or an Elf-1 antagonist, in order to promote orinhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homolog of limited function,and with fewer side effects relative to treatment with agonists orantagonists which are directed to all of the biological activities ofnaturally occuring forms of the Elf-1 protein. For instance, Elf-1homologs can be generated which interfere with the ability of thewild-type protein in forming complexes with either the mek4 or sekreceptor proteins, but which do not substantially interfere with theformation of complexes between the Elf-1 polypeptide and other membersof the EPH receptor family, such as may be involved in other signaltransduction mechanisms.

Homologs of the subject Elf-1 protein can be generated by mutagenesis,such as by discrete point mutation(s) or by truncation. For instance,mutation can give rise to homologs which retain substantially the same,or merely a subset, of the biological activity of the Elf-1 polypeptidefrom which it was derived. Alternatively, antagonistic forms of theprotein can be generated which are able to inhibit the function of thenaturally occurring form of the protein, such as by competitivelybinding to an EPH receptor, e.g. mek4 or sek.

A protein has Elf-1 polypeptide biological activity if it has one ormore of the following properties: the ability to modulate proliferation,survival and/or differentiation of a cell which expresses an EPHreceptor, such as a hek-related or sek-related receptor, the ability tomodulate proliferation, survival and/or differentiation ofmesodermally-derived tissue, such as tissue derived from dorsalmesoderm; the ability to modulate proliferation, survival and/ordifferentiation of ectodermally-derived tissue, such as tissue derivedfrom the neural tube, neural crest, or head mesenchyme; the ability tomodulate proliferation, survival and/or differentiation ofendodermally-derived tissue, such as tissue derived from the primitivegut. In general, the ability to bind an EPH receptor protein, e.(g. a,7nek4, hek, tyro4, cek4, sek, cek8, or tyro1 receptor, is sufficient tocharacterize the polypeptide as an Elf-1 polypeptide of the presentinvention. Thus, according to the present invention, a polypeptide hasbiological activity if it is a specific agonist or antagonist of anaturally-occurring form of a Elf-1 protein.

Preferred nucleic acids encode an Elf-1 polypeptide comprising an aminoacid sequence at least 60% homologous, more preferably 70% homologousand most preferably 80% homologous with an amino acid sequence shown inone of SEQ ID No: 1. Nucleic acids which encode polypeptides having anactivity of an Elf-1 polypeptide and having at least about 90%, morepreferably at least about 95%, and most preferably at least about 98-99%homology with a sequence shown in one of SEQ ID No: 1 are also withinthe scope of the invention. In one embodiment, the nucleic acid is acDNA encoding a peptide having at least one activity of the subjectElf-1 polypeptide. Preferably, the nucleic acid is a cDNA moleculecomprising at least a portion of the nucleotide sequence represented inSEQ ID No: 1. A preferred portion of this cDNA molecules includes thecoding region of the gene.

Another aspect of the invention provides a nucleic acid which hybridizesunder high or low stringency conditions to a nucleic acid which encodesa peptide having all or a portion of an amino acid sequence shown in SEQID No: 1. Appropriate stringency conditions which promote DNAhybridization, for example, 6.0×sodium chloride/sodium citrate (SSC) atabout 45° C., followed by a wash of 2.0×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (I 989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C.

Nucleic acids, having a sequence that differs from the nucleotidesequence shown in SEQ ID No: 1 due to degeneracy in the genetic code arcalso within the scope of the invention, Such nucleic acids encodefunctionally equivalent peptides (i.e., a peptide havinlg a biologicalactivity of, Elf-1 polypeptide) but differ in sequence from the sequenceshown in the sequence listing due to degeneracy in the genetic code. Forexample, a number of amino acids are designated by more than onetriplet. Codons that specify the same amino acid, or synonyms (forexample, CAU and CAC each encode histidine) may result in “silent”mutations which do not affect the amino acid sequence of the Elf-1polypeptide. However, it is expected that DNA sequence polymorphismsthat do lead to changes in the amino acid sequences of the subject Elf-1polypeptides will exist among vertebrates. One skilled in the art willappreciate that these variations in one or more nucleotides (up to about3-5% of the nucleotides) of the nucleic acids encoding polypeptideshaving an activity of an Elf-1 polypeptide may exist among individualsof a given species due to natural allelic variation. Any and all suchnucleotide variations and resulting amino acid polymorphisms are withinthe scope of this invention.

Fragments of the nucleic acids encoding an active portion of the Elf-1protein are also within the scope of the invention. As used herein, afragment of the nucleic acid encoding the active portion of an Elf-1polypeptide refers to a nucleic acid having fewer nucleotides than thenucleotide sequence encoding the entire amino acid sequence of the Elf-1protein represented in SEQ ID No: 2, but which nevertheless encodes apeptide having an Elf-1 polypeptide biological activity, e.g. thefragment retains the ability to bind to an EPH receptor such as mek4 orsek. For instance, Elf-1 polypeptides can provided which lack anendogenous signal sequence or a phosphatidylinositol attachcment site.Nucleic acid fragments within the scope of the present invention includethose capable of hybridizing under high or low stringency conditionswith nucleic acids from other species for use in screening protocols todetect Elf-1 homologys, as well as those capable of hybridizing withnucleic acids from human specimens for use in detecting the presence ofa nucleic acid encoding the subject Elf-1 protein, including alternateisoforms. e.g. mRNA splicing variants. Nucleic acids within the scope ofthe invention may also contain linker sequences, modified restrictionendonuclease sites and other sequences useful for molecular cloning,expression or purification of recombinant forms of the subject Elf-1polypeptides.

As indicated by he examples set out below, a nucleic acid encoding apeptide having an activity of an Elf-1 polypeptide may be obtained frommRNA present in any of a number of eukaryotic cells. It should also bepossible to obtain nucleic acids encoding Elf-1 polypeptides of thepresent invention from genomic DNA obtained from both adults andembryos. For example, a gene encoding an Elf-1 protein can be clonedfrom either a cDNA or a genomic library in accordance with protocolsdescribed herein, as well as those generally known to persons skilled inthe art. A cDNA encoding an Elf-1 protein can be obtained by isolatingtotal mRNA from a cell, e.g. a mammalian cell, e.g. a human cell,including embryonic cells. Double stranded cDNAs can then be preparedfrom the total mRNA, and subsequently inserted into a suitable plasmidor bacteriophage vector using any one of a number of known techniques.The gene encoding the Elf-1 protein can also be cloned using,established polymerase chain reaction techniques in accordance with thenucleotide sequence information provided by the invention. The nucleicacid of the invention can be DNA or RNA. A preferred nucleic acid is acDNA represented by the sequence shown in SEQ ID No: 1.

Another aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, “antisense” therapyrefers to administration or in situ generation of oligonucleotide probesor their derivatives which specifically hybridizes (e.g. binds) undercellular conditions, with the cellular mRNA and/or genomic DNA encodingan Elf-1 protein so as to inhibit expression of that protein, e.g. byinhibiting transcription and/or translation. The binding may be byconventional base pair complementarity, or, for example, in the case ofbinding to DNA duplexes, through specific interactions in the majorgroove of the double helix. In general, “antisense” therapy refers tothe range of techniques generally employed in the art, and includes anytherapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes an Elf-1 protein. Alternatively, theantisense construct is an oligonucleotide probe which is generated exvivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of amElf-1 (gene. Such oligonucleotide probes are preferably modifiedoligonucleotide which are resistant to endogenous nucleases, e.g.exonucleases and/or endonucleases, and is therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphoiiate analogs ofDNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example by van der krol et al.(1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668.

Accordingly, the modified oligomers of the invention are useful intherapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general. For such therapy, the oligomers of theinvention can be formulated for a variety of loads of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. For systemic administration,injection is preferred, including intramuscular, intravenous,intraperitoneal, and subcutaneuos for injection, the oligomers of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligomers may be formulated in solid form andredissolved or suspended immediately prior to use Lyophilized forms arealso included.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration. penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration bile salts and fusidic acid derivatives. In addition,detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays or using suppositories. Fororal administration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics. For topicaladministration, the oligomers of the invention are formulated intoointments, salves, gels, or creams as generally known in the art.

In addition to use in therapy, the oligomers of the invention may beused as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are described in further detail below.

Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of Elf-1, can be used in themanipulation of tissue, e.g. tissue differentiation, both in vivo and inex vivo tissue cultures.

This invention also provides expression vectors containing a nucleicacid encoding an Elf-1 polypeptide, operably linked to at least onetranscriptional regulatory sequence. Operably linked is intended to meanthat the nucleotide sequence is linked to a regulatory sequence in amanner which allows expression of the nucleotide sequence. Regulatorysequences are art-recognized and arc selected to direct expression ofthe subject Elf-1 proteins. Accordingly, the term transcriptionalregulatory sequence includes promoters, enhancers and other expressioncontrol elements. Such regulatory sequences are described in Goeddel;Gene Expression Technology: Methods in Enzymoloy 185. Academic Press,San Diego, Calif. (1990). For instance, any of a wide variety ofexpression control sequences-sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding the Elf-1 polypeptides of thisinvention. Such useful expression control sequences, include, forexample, a viral LTR, such as the LTR of the Moloney murine leukemiavirus, the early and late promoters of SV40, adenovirus orcytomegalovirus immediate early promoter. the lac system, the trpsystem, the TAC or TRC system, T7 promoter whose expression is directedby T7 RNA polymerase, the major operator and promoter regions of phageλ, the control regions for fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Moreover, the vector's copynumber, the ability to control that copy number and the expression ofany other proteins encoded by the vector, such as antibiotic markers,should also be considered. In one embodiment, the expression vectorincludes a recombinant gene encoding a peptide having an agonisticactivity of a subject Elf-1 polypeptide, or alternatively, encoding apeptide which is an antagonistic form of the Elf-1 protein. Suchexpression vectors can be used to transfect cells and thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

Moreover, the gene constructs of the present invention can also be usedas a part of a gene therapy protocol to deliver nucleic acids encodingeither an agonistic or antagonistic form of the subject Elf-1 protein.Thus, another aspect of the invention features expression vectors for invivo transfection and expression of an Elf-1 polypeptide in particularcell types so as to reconstitute the function of, or alternatively,abrogate the function of Elf-1 in a tissue in which Elf-1 ismisexpressed; or to deliver a form of the protein which altersdifferentiation of tissue, or which inhibits neoplastic transformation,by modulating the biological function of an EPH receptor (e.g. the mek4or sek receptors).

Expression constructs of the subject Elf-1 polypeptide, and mutantsthereof, may be administered in any biologically effective carrier, e.g.any formulation or composition capable of effectively delivering theElf-1 gene to cells in vivo. Approaches include insertion of the subjectgene in viral vectors including recombinant retroviruses. adenovirus,adeno-associated virus, and herpes simplex virus-1, or recombinantbacterial or eukaryotic plasmids. Viral vectors transfect cellsdirectly; plasmid DNA can be delivered with the help of, for example,cationic liposomes (lipofectin) or derivatized (e.g. antibodyconjugated), polylysine conjugates, gramacidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo. It will be appreciated that because transduction of appropriatetarget cells represents the critical first step in gene therapy, choiceof the particular gene delivery system will depend on such factors asthe phenotype of the intended target and the route of administration,e.g. locally or systemically. Furthermore, it will be recognized thatthe particular gene construct provided for in vivo transduction of Elf-1expression are also useful for in vitro transduction of cells, such asfor use in the ex vivo tissue culture systems described below.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA,encoding the particular form of the Elf-1 polypeptide desired. Infectionof cells with a viral vector has the advantage that a large proportionof the targeted cells can receive the nucleic acid. Additionally,molecules encoded within the viral vector, e.g., by a cDNA contained inthe viral vector, are expressed efficicntly in cells which have taken upviral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termiied “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfel for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding oneof the subject receptors rendering the retrovirus replication defective.The replication defective retrovirus is then packaged into virions whichcan be used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines for preparing bothecotropic and amphotropic retroviral systems include ψCrip, ψCre, ψ2 andψAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including neuronal cells, in vitro and/or invivo (see for example Eglitis, et al. (1985) Science 230:1395-1398;Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464;Wilson et al. (1988) Proc. Natl. Acad. USA 85:3014-3018; Armentano etal. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991)Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (199i) Proc. Natl.Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acaid. Sci. USA89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al.(1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J.Immunol. 150:4104-4115: U.S. Pat. No. 4,868,116; U.S. Pat. No.4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum ol retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J. BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (e.g. lactose to convertthe env protein to an asialoglycoprotein), as well as by generatingfusion proteins (e.(g. single-chain antibody/env; fusion proteins). Thistechnique, while useful to limit or otherwise direct the infection tocertain tissue types, can also be used to convert an ecotropic vector into an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue-or cell-specific transcriptional regulatory sequenceswhich control expression of the Elf-1 gene of the retroviral vector.

Another viral gene delivery system useful in the present inventionutilitizes adenovirus-derived vectors. The genome of an adenovirus canbe manipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniqtues 6:616; Rosenfeld et al. (1991) Science 252:431-434: andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses can be advantageous in certaincircumstances in that they are not capable of infecting nondividingcells and can be used to infect a wide variety of cell types, includingepithelial cells (Rosenfeld et al. (1992) cited supra) Furthermore, thevirus particle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in sitilations whereintroduced DNA becomes integrated into the host genome (e.g., retroviralDNA). Morcover, the carrying capacity of the adenoviral genome forforeign DNA is large (up to 8 kilobases) relative to other gene deliveryvectors (Berkner et al. cited supra: Haj-Ahmand and Graham (1986) J.Virol. 57:267). Most replication-defective adenoviral vectors currentlyin use and therefore favored by the present invention are deleted forall or parts of the viral E1 and E3 genes but retain as much as 80% ofthe adenoviral genetic material (see, e.g., Jones et al. (1979) Cell16:683: Berkner et al., supra; and Graham et al. in Methods in MolecularBiology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp.109-127). Expression of the inserted Elf-1 gene can be under control of,for example, the E1A promoter, the major late promoter (MLP) andassociated leader sequences, the E3 promoter, or exogenously addedpromoter sequences.

Yet another viral vector system useful for delivery of the subject Elf-1gene is the adeno-associated virus (AAV). Adeno-associated virus is anaturally occurring defective virus that requires another virus, such asan adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can intc()rate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081: Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chemn. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of an Elf-1polypeptide in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the subject Elf-1polypeptide gene by the targeted cell. Exemplary gene delivery systemsof this type include liposomal derived systems, poly-lysine conjugates,and artificial viral envelopes.

In a representative embodiment, a gene encoding one of the subject Elf-1polypeptides can be entrapped in liposomes bearing positive charges ontheir surface (e.g., lipofectins) and (optionally) which are tagged withantibodies against cell surface antigens of the target tissue (Mizuno etal. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309;Japanese patent application 1047381; and European patent publicationEP-A-43075). For example, lipofection of cells can be carried out usingliposomes tagged with monoclonal antibodies against any cell surfaceantigen present on the tumor cells, as for example, the CD20 antigenwhich has been detected on the lymphoblastic cell line LK63/CD20+ whichalso expresses the hek receptor (Wicks et al. (1992) PNAS 89:1611-1615).

In clinical settings, the gene delivery systems for the therapeuticElf-1 gene can be introduced into a patient by any of a number ofmethods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g. by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994)-PNAS 91: 3054-3057).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

Another aspect of the present invention concerns recombinant forms ofthe subject Elf-1 protein which are encoded by genes derived fromeukaryotic organisms such as mammals, e.g. humans. Recombinant proteinspreferred by the present invention, in addition to native Elf-1polypeptides. are at least 60% homologous, more preferably 70%homologous and most preferably 80% homologous with an amino acidsequence shown in SEQ ID No: 1. Polypeptides having an activity of thesubject Elf-1 polypeptides (i.e. either agonistic or antagonistic) andhaving at least about 90%, more preferably at least about 95%, and mostpreferably at least about 98-99% homology with a sequence in SEQ ID No:1 are also within the scope of the invention.

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally DNA encoding an Elf-1 polypeptide is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the heterologous protein. Moreover, the phrase “derived from”with respect to a recombinant Elf-1 gene, is meant to include within themeaning of “recombinant protein” those proteins having an amino acidsequence of a native Elf-1 polypeptide, or an amino acid sequencesimilar thereto which is generated by mutations including substitutionsand deletions (including truncation) of a naturally occurring form of anElf-1 protein. For instance, N-glycosylation sites in the Elf-1 proteincan be modified (e.g. mutated) to preclude glycosylation, allowingexpression of a more homogenous, reduced carbohydrate analog inmammalian, insect and yeast expression systems. The wild-type proteincontains three potential N-linked glycosylation sites which can bemutated: Asn-38, Asn-170 and Asn-184. likewise, Elf-1 polypeptides canbe generated which lack an endogenous signal sequence (though this istypically cleaved off even if present in the pro-form of the protein),or which lack a phosphatidylinositol linkage site to preclude additionof phosphatidylinositol. In the instance of the latter, the removal ofthe C-terminus may result in a soluble form of the protein. Inparticular. polypeptides which lack at least the last 15 amino acidresidues (truncated at Leu-195) are preferred as soluble forms of theprotein, though polypeptides which are truncated any where betweenThr-182 to Leu-195 are also contemplated.

Furthermore, comparison of the subject protein with the only two otherEPH receptor ligands known, namely B61 (Bartley et al. (1994) Nature368:558-561: and Holzman et al. (1990) Mol. Cell Biol 10:5830-5838) andLERK-2 (Beckmann et al. (1994) EMJO J13:3757-3762; PCT PublicationWO95/11384), suggests that the biological activity of the moleculeresides largely in the region of the protein containing the fourcysteines whose spacing is apparently conserved so as to suggest animportant motif. Consequently, a preferred Elf-1 polypeptide comprisesCys-69 through Cys-159, or a sequence homologous thereto.

The present invention further pertains to recombinant forms of thesubiject Elf-1 polypeptides which are encoded by genes derived from avertebrate organism, particularly a mammal (e.g. a human), and whichhave amino acid sequences evolutionarily related to the Elf-1 proteinrepresented in SEQ ID No: 1. Such recombinant Elf-1 polypeptidespreferably are capable of functioning, in one of either role of anagonist or antagonist of at least one biological activity of the Elf-1polypeptide of the appended sequence listing. The term “evolutionarilyrelated to”, with respect to amino acid sequences of the presentrecombinant Elf-1 polypeptides, refers to Elf-1 polypeptides havingamino acid sequences which have arisen naturally, and also to mutationalvariants of Elf-1 polypeptides which are derived, for example, bycombinatorial mutagenesis. Such evolutionarily derived Elf-1polypeptides preferred by the present invention are at least 60%homologous, more preferably 70% homologous and most preferably 80%homologous with the amino acid sequence shown in SEQ ID No: 1.Polypeptides having at least about 90%, more preferably at least about95%, and most preferably at least about 98-99% homology with a sequenceshown in SEQ ID No: 1 are also within the scope of the invention.

The present invention further pertains to methods of producing thesubject Elf-1 polypeptides. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the subject Elf-1 polypeptide can be cultured under appropriatecondi tons to allow expression of the peptide to occur. The peptide maybe secreted and isolated from a mixture of cells and medium containingthe recombinant Elf-1 polypeptidc. Alternatively, the peptide may beretained cytoplasmically by removing the signal peptide sequence fromthe recombinant Elf-1 gene and the cells harvested, lysed and theprotein isolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The recombinant Elf-1 polypeptide peptide can be isolated from cellculture medium, host cells, or both using techniques known in the artfor purifying proteins including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for such peptide.In a preferred embodiment, the recombinant Elf-1 polypeptide is a fusionprotein containing a domain which facilitates its purification, such asan Elf-1/GST fusion protein.

This invention also pertains to a host cell transfected to express arecombinant form of the subject Elf-1 polypeptides. The host cell may beany prokaryotic or eukaryotic cell, and the choice can be based at leastin part on the desirablity of such post-translation modifications asglycosylation and/or addition of phosphatidylinositol. Thus, anucleotide sequence derived from the cloning of Elf-1, encoding all or aselected portion of the full-length protein, can be used to produce arecombinant form of an Elf-1 polypeptide via microbial or cukaryoticcellular processes. Ligating the polynucleotide sequence into a geneconstruct, such as an expression vector, and transforming ortransfecting into hosts, either eukaryotic (yeast, avian, insect ormammalian) or prokaryotic (bacterial cells), are standard proceduresused in producing other well-known proteins, e.g. insulin, interferons,human growth hormone, IL-1, IL-2, and the like. Similar procedures, ormodifications thereof, can be employed to prepare recombinant Elf-1polypeptides by microbial means or tissue-culture technology in accordwith the subject invention.

The recombinant Elf-1 gene can be produced by ligating nucleic acidencoding the subject Elf-1 protein, or a portion thereof into a vectorsuitable for expression in either prokaryotic cells, eukaryotic cells,or both. Expression vectors for production of recombinant forms of thesubject Elf-1 polypeptides include plasmids and other vectors. Forinstance, suitable vectors for the expression of an Elf-1 polypeptideinclude plasmids of the types: pBR322-derived plasmids, pEMBL,-derivedplasmids, pEX-derived plasmids. pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YFP24, YIP5. YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach el al. (1983) inExperimenial Manipulcition of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, an Elf-1 polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of the Elf-1 gene represented in SEQ ID NO. 1.

The preferred mammalian expression vectors contain both prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukarvotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaborolory Manual. 2nd Ed., ed. by Sambrook. Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17. In someinstances, it may be desirable to express the recombinant Elf-1polypeptide by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL,1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBacIII).

When it is desirable to express only a portion of an Elf-1 protein, suchas a form lacking a portion of the N-terminus, i.e. a trunction mutantwhich lacks the signal peptide, it may be necessary to add a start codon(ATG) to the oligonucleotide fragment containing the desired sequence tobe expressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al. (1987) J. Bacteriol. 169:751-XX57) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing Elf-1-derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al.,supra).

Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. This type of expression system can beuseful under conditions where it is desirable to produce an immunogenicfragment of an Elf-1 protein. For example, the VP6 capsid protein ofrotavirus can be used as an immunologic carrier protein for portions ofthe Elf-1 polypeptide, either in the monomeric form or in the form of aviral particle. The nucleic acid sequences corresponding to the portionof a subject Elf-1 protein to which antibodies are to be raised can beincorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising Elf-1epitopes as part of the virion. It has been demonstrated with the use ofimmunogenic fusion proteins utilizing the Hepatitis B surface antigenfusion proteins that recombinant Hepatitis B virions can be utilized inthis role as well. Similarly chimeric constructs coding for fusionproteins containing a portion of an Elf-1 protein and the polioviruscapsid protein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see, for example, EP Publication No: 0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

The Multiple Antigen Peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofan Elf-1 polypeptide is obtained directly from organo-chemical synthesisof the peptide onto an oligomeric branching lysine core (see, forexample, Posnett et al. (1988) JBC 263: 1719 and Nardelli et al. (1992)J. Immunol. 148:914). Antigenic determinants of Elf-1 proteins can alsobe expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, including the Elf-1 polypeptides of the presentinvention. For example, an Elf-1 polypeptide can be generated as aglutathione-S-transferase (GST-fusion protein). Such GST-fusion proteinscan enable easy purification of the Elf-1 polypeptide, as for example bythe use of glutathione-derivatized matrices (see, for example, CurrentProtocols in Moleculcar Biology, eds. Ausubel et al. (N.Y.: John Wiley &Sons, 1991)). In another embodiment, a fusion gene coding for apurification leader sequence, such as a poly-(His)/enterokinase cleavagesite sequence, can be used to replace the signal sequence whichnaturally occurs at N-terminus the Elf-1 protein, in order to permitpurification of the poly(His)-Elf-1 protein by aflinity chromatographyusing a Ni²⁺ metal resin. The purification leader sequence can then besubsequently removed by treatment with enterokinase (e.g., see Hochuliet al. (1987) J. Chromatography 411:177; and Janknecht et al. PNAS88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a ehimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

The Elf-1 polypeptide may also be chemically modified to create Elf-1derivatives by forming covalent or aggregrative conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of Elf-1 can be prepared bylinking the chemical mocities to functional groups on Elf-1 amino acidsidechains or at the N-terminus or at the C-terminus of the polypeptide.For instance, an Elf-1 protein can generated which includes a moiety,other than sequences naturally associated with the Elf-1 protein, thatbinds a component of the extracellular matrix and enhances localizationof the Elf-1 analog to cell surfaces. For example, sequences derivedfrom the fibronectin “type-III repeat”, such as a tetrapeptide sequenceR-G-D-S (Pierschbacher et al. (1984) Nature 309:30-3; and Kornblihtt etal. (1985) EMBO 4:1755-9) can be added to the Elf-1 polypepyide tosupport attachment of the chimeric molecule to a cell through bindingECM components (Ruoslahti et al. (1987) Science 238:491-497;Pierschbacheret al. (1987) J. Biol. Chem. 262:17294-8.; Hynes (1987)Cell 48:549-54; and Hynes (1992) Cell 69:11-25) particularly where theElf-1 polypeptide lacks a C-terminal phosphatidylinositol.

The present invention also makes available isolated Elf-1 polypeptideswhich are isolated from, or otherwise substantially free of othercellular and extracellular proteins, especially EPH receptor proteins orother extracellular factors, normally associated with the Elf-1polypeptide. The term “substantially free of other cellular orextracellular proteins” (also referred to herein as “contaminatingproteins”) or “substantially pure or purified preparations” are definedas encompassing preparations of Elf-1 polypeptides having less than 20%(by dry weight) contaminating protein, and preferably having less than5% contaminating protein. Functional forms of the subject Elf-1polypeptides can be prepared. for the first time, as purifiedpreparations by using a cloned gcnle as described herein. Alternatively,the subject Elf-1 polypeptides can be isolated by affinity purificationusing, for example, matrix bound EPH receptor protein. By “purified”, itis meant, when referring to a peptide or DNA or RNA sequence, that theindicated molecule is present in the substantial absence of otherbiological macromolecules, such as other proteins. Te term “purified” asused herein preferably means at least 80% by dry weight, more preferablyin the range of 95-99% by weight, and most preferably at least 99.8% byweight, of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). The term “pure” asused herein preferably has the same numerical limits as “purified”immediately above. “Isolated” and “purified” do not encompass eithernatural materials in their native state or natural materials that havebeen separated into components (e.g., in an acrylamide gel) but notobtained either as pure (e.g. lacking contaminating proteins, orchromatography reagents such as denaturing agents and polymers, e.g.acrylamide or agarose) substances or solutions.

As described above for recombinant polypeptidcs, isolated Elf-1polypeptides can include all or a portion of the amino acid sequencerepresented in SEQ ID No. 2, or a homologous sequence thereto. Exemplaryderivatives of that sequence include proteins which lack N-glycosylationsites (e.g. to produce an unglycosylated protein), lack an N-terminusand or/C-terminus sequence from SEQ ID No. 2, e.g. an Elf-1 polypeptidewhich comprises Cys-69 through Cys-159, or a sequence homologousthereto.

Furthermore, isolated peptidyl portions of Elf-1 proteins can also beobtained by screening peptides recombinantly produced from thecorresponding fragment of the nucleic acid encoding such peptides. Inaddition, fragments can be chemically synthesized using techniques knownin the art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, an Elf-1 polypeptide of the present inventionmay be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as either agonists or antagonistsof an Elf-1 polypeptide activity, such as by in vivo competition assaysor in vitro protein binding assays with EPH receptors.

It will also be possible to modify the structure of the subject Elf-1polypeptides for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified peptides, when designedto retain at least one activity of the naturally-occurring form of theprotein, are considered functional equivalents of the Elf-1 polypeptidedescribed in more detail herein. Such modified peptide can be produced,for instance, by amino acid substitution, deletion, or addition.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. conservative mutations) will nothave a major effect on the biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Geneticallyencoded amino acids are can be divided into four families: (1)acidic=aspartate, glutamate; (2) basic=lysine, argininie, histidine, (3)nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar=glycine, asparagine,glutainine, cysteine, serine, threonine, tyrosine. Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In similar fashion, the amino acid repertoire can begrouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, argininehistidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine,serine, threonine, with serine and threonine optionally be groupedseparately as aliphatic-hydroxyl, (4) aromatic=phenylalanine, tyrosine,tryptophan; (5) amide=asparagine, glutamine; and (6)sulfur-containing=cysteine and methionine. (see, for example,Biochemistry, 2nd ed., Ed. by L. Stryer, WH Freeman and Co.: 1981).Whether a change in the amino acid sequence of a peptide results in afunctional Elf-1 homolog (e.g. functional in the sense that it acts tomimic or antagonize the wild-type form) can be readily determined byassessing the ability of the variant peptide to produce a response incells in a fashion similar to the wild-type Elf-1 protein orcompetitively inhibit such a response. Peptides in which more than onereplacement has taken place can readily be tested in the same manner.

Accordingly, the present invention contemplates a method of generatingsets of combinatorial mutants of the presently disclosed novel Elf-1polypeptides, as well as truncation and fragmentation mutants, and isespecially useful for identifying potential variant sequences which arefunctional in binding to an EPH receptor. One purpose for screening suchcombinatorial libraries is, for example, to isolate novel Elf-1 homologswhich function as one of either an agonist or antagonist of thebiological activities of the wild-type (“authentic”) protein, oralternatively, which possess novel activities all together. Toillustrate, Elf-1 homologs can be engineered by the present method toprovide proteins which bind an EPH receptor, such as mek4/cek4/hek/tyro4or sek/cek8/tyro1 receptors, yet which block receptor-mediated genetranscription resulting from signal transduction pathways normallyassociated with activation of that receptor. Such proteins, whenexpressed from recombinant DNA constructs, can be used in gene therapyprotocols, or can be formulated as pharmaceutical preparations anddelivered in such manner.

Likewise, mutagenesis can give rise to Elf-1 homologs which haveextracellular half-lives dramatically different than the correspondingwild-type protein. For example, the altered protein can be renderedeither more stable or less stable to proteolytic degradation or otherextracellular process which result in destruction of, or otherwiseinactivation of, an Elf-1 polypeptide. Such Elf1I homologs can beutilized to alter the envelope of bioavailabilty for a recombinant Elf-1protein by modulating, for example, the plasma half-life of the protein.For instance, a short half-life can give rise to more transientbiological effects associated with a particular recombinant Elf-1polypeptidc and can therefore allow tighter control of protein levelswithin or around a particular tissue. As above, such proteins. andparticularly their recombinant nucleic acid constructs, can be used ingene therapy protocols as well as formulated into pharmaceuticalpreparations.

In an illustrative embodiment of this method, the amino acid sequencesfor a population of Elf-1 homologs or other related proteins arealigned. preferably to promote the highest homology possible. Such apopulation of variants can include, for example, Elf-1 homologs from oneor more species, e.g. murine and human, or Elf-1 homologs from the samespecies but which differ due to mutation. Amino acids which appear ateach position of the aligned sequences are selected to create adegenerate set of combinatorial sequences. There are many ways by whichthe library of potential Elf-1 homologs can be generated from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be carried out in an automatic DNA synthesizer, andthe synthetic genes then be ligated into an appropriate gene forexpression. The purpose of a degenerate set of genes is to provide, inone mixture, all of the sequences encoding the desired set of potentialElf-1 polypeptide sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, SA(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Synmpos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierpp. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakuraet al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.11:477. Such techniques have been employed in the directed evolution ofother binding proteins (see, for example, Scott et al. (1990) Science249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al.(1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; aswell as U.S. Pat. Nos: 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library, particularly where no other naturally occurringhomologs have yet been sequenced. For example, Elf-1 homologs (bothagonist and antagonist forms) can be generated and isolated from alibrary bv screening, using, for example, alanine scanning mutagenesisand the like (Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al.(1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601;Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892: Lowman et al.(1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science244:1081-1085), by linker scanning mutagenesis (Gustin et al. (1993)Virology 193:653-660; Brown et al. (1992) Mol. Cell Biol. 12:2644-2652;McKnight et al. (1982) Science 232:316); by saturation mutagenesis(Meyers et al. (1986) Science 232:613); by PCR mutagenesis (Leung et al.(1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis (Milleret al. (1992) A Short Course in Bacterial Genetics, CSHL Press, ColdSpring Harbor, N.Y.; and Greener et al. (1994) Strategies in Mol Biol7:32-34).

A wide range of techniques are known in the art for screening, geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis of Elf-1polypeptides. The most widely used techniques for screening large genelibraries typically comprises cloning the gene library into rcplicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected. Each of the illustrative assays described below areamenable to high through-put analysis as necessary to screen largenumbers of degenerate Elf-1 sequences created by combinatorialmutagenesis techniques.

In one screening assay, the candidate Elf-1 polypeptides are displayedon the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to bind an EPH receptor protein viathis gene product is detected in a “panning assay”. For instance, thegene library can be cloned into the gene for a surface membrane proteinof a bacterial cell, and the resulting fusion protein detected bypanning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology9:1370-1371; and Goward et al. (1992) TIBS 18:136-140). In a similarfashion, a detectably labeled HPH receptor can be used to score forpotentially functional Elf-1 polypeptide homologs. For example, theAP-mek4 or Ap-sek fusion proteins described below, or the equivalentfluorcscently labeled receptors. can be used to detect Elf-1 homologwhich retain reccptor-binding activity. In the application offluorescently labeled receptor, cells can be visually inspected andseparated under a fluorescence microscope, or, where the morphology ofthe cell permits, separated by a fluorescence-activated cell soiter.

In an alternate embodiment, the gene library is expressed as a fusionprotein on the surface of a viral particle. For instance, in thefilamentous phage system, forei(gn peptide sequences can be expressed onthe surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, a large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd., and fl are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02909: Garrardet al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992)PNAS89:4457-4461). For example, the recombinant phage antibody system(RPAS, Pharmacia Catalog number 27-9400-01) can be easily modified foruse in expressing and screening Elf-1 combinatorial libraries by panningon glutathione immobilized EPH receptor/GST fusion proteins to enrichfor Elf-1 homologs which retain an ability to bind an EPH receptor.

Each of these homologs can subsequently be screened for furtherbiological activities in order to differentiate agonists andantagonists. For example, receptor-binding homologs isolated from thecombinatorial library can be tested for their effect on cellularproliferation relative to the wild-type form of the protein.Alternatively, one could screen the homologs for agonists by detectingautophosphorylation of an EPH receptor in response to treatment with thehomolog (see, for example, Millauer et al. (1993) Cell 72:835-846). Insimilar fashion, antagonists can be identified from the enrichedfraction based on their ability to inhibit autophosphorylationof thereceptor by wild-type Elf-1 protein.

In another embodiment, the combinatorial library is designed to beextracellularly presented (e.g. as it occurs naturally) and, thoughoptionally, secreted (e.g. the polypeptides of the library all include asignal sequence but no phosphatidylinositol linkage). The gene can beused to transfect a eukaryotic cell that can be co-cultured with cellswhich express an functional EPH receptor, e.g. a hek-related receptor,and which are sensitive to treatement with the wild-type soluble form ofElf-1. Functional Elf-1 homologs secreted by the cells expressing thecombinatorial library will diffuse to neighboring EPH+cells and induce aphenotypic change. Using, for example, antibodies directed to epitopeswhich are either created or destroyed in response to Elf-1 treatment,the pattern of detection of Elf-1 induction will resemble a gradientfunction, and will allow the isolation (generally after severalrepetitive rounds of selection) of cells producing active Elf-1homologs. Likewise, Elf-1 antagonists can be selected in similar fashionby the ability of the cell producing a functional antagonist to protectneighboring cells from the effect of authentic Elf-1 added to theculture media.

To illustrate, target cells are cultured in 24-well microtitre plates.The target cells can be, for instance, cells which naturally expresshek-related or sek-related receptors, such as NIH 3T3 cells, or cellswhich have been transfected with genes encoding such a receptor. COScells are transfected with the combinatorial Elf-1 gene library andcultured (optionally) in a cell culture insert (e.g. CollaborativeBiomcdical Products, Catalog #40446) that are able to fit into the wellsof the microtitre plate. The cell culture inserts are placed in thewells such that recombinant Elf-1 homologs secreted by the cells in theinsert can diffuse through the porous bottom of the insert and contactthe target cells in the microtitre plate wells. After a period of timesufficient for functional forms of Elf-1 to produce a measurableresponse in the target cells. the inserts are removed and the effect ofany Elf-1 homologs on the target cells determined. Cells from theinserts corresponding to wells which score positive for activity can besplit and re-cultured on several inserts, the process being repeateduntil the active clones are identified.

The invention also provides for reduction of the Elf-1 protein togenerate mimetics, e.g. peptide or non-peptide agents, which are able todisrupt binding of an Elf-1 polypeptide of the present invention with anEPH receptor. Thus, such mutagenic techniques as described above arealso useful to map the determinants of the Elf-1 polypeptide whichparticipate in protein-protein interactions involved in, for example,binding of the subject Elf-1 polypeptide to an EPH receptor or incausing oligomcrization of receptors. To illustrate, the criticalresidues of a subject Elf-1 polypeptide which are involved in molecularrecognition of an EPH receptor can be deternined and used to generateElf-1 polypeptide-derived peptidomimeties which competitively inhibitbinding of the authentic Elf-1 protein with that receptor. By employing,for example, scanning mutagenesis to map the amino acid residues of theElf-1 protein involved in binding the mek4 receptor, peptidomimeticcompounds can be generated which mimic those residues in binding to thereceptor and which consequently can inhibit binding of Elf-1 to the mek4receptor and interfere with the function of the mek4 receptor. IForinstance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Hufffman et al. in Peptides.Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gama lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagai et al.(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc PerkinTrans 1:1231), and β-aminoalcohols (Gordon et al. (1985) BiochemBiolphys Res Commun 26:419; and Danl et al. (1986) Biochem Biophys ResCommun 134:71).

Another aspect of the invention pertains to an antibody specificallyreactive with an Elf-1 protein. For example, by using immunogens derivedfrom the Elf-1 protein, e.g. based on the cDNA sequences,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols (See, for example, Antibodies: A Laboratory Manualed. by Harlow and lane (Cold Spring Harbor Press: 1988)). A mammal, suchas a muse, a hamster or rabbit can be immunized with an immunogenic formof the peptide (e.g. an Elf-1 polypeptide or an antigenic fragment whichis capable of eliciting an antibody response). Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques well known in the art. An immunogenic portion of theElf-1 protein can be administered in the presence of adjuvant. Theprogress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immuinoassays can beused with the immunogen as antigen to assess the levels of antibodies.In a preferred embodiment, the subject antibodies are immunospecitic forantigenic determinants of the Elf-1 protein of the present invention,e.g. antigenic determinants of a protein represented by SEQ ID No: 1 ora closely related human or non-human mammalian homolog (e.g. at least 85percent homologous, preferably at least 90 percent homologous, and morepreferably at least 95 percent homologous). In yet a further preferredembodiment of the present invention, the anti-Elf-1 polypeptideantibodies do not substantially cross react (i.e. does not reactspecifically) with a protein which is, for example, less than 85 percenthomologous to SEQ ID No: 1; e.g. less than 95 percent homologous withone of SEQ ID No: 1; e.g. less than 98-99 percent homologous with one ofSEQ ID No: 1. By “not substantially cross react”. it is meant that theantibody has a binding affinity for a non-homologous protein (e.g.LERK-2 or the B61 protein) which is at least one order of magnitude morepreferably at least 2 orders of magnitude, and even more preferably atleast 3 orders of magnitude less than the binding affinity of theantibody for the protein of SEQ ID No: 1.

Following immunization, anti-Elf-1 antisera can be obtained and, ifdesired, polyclonal anti-Elf-1 antibodics isolated from the serum. Toproduce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc, pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an Elf-1 polypeptideof the present invention and monoclonal antibodies isolated from aculture comprising such hybridoma cells.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectElf-1 polypeptide. Antibodies can be fragmented using, conventionaltechniques and the fragments screened for utility in the same manner asdescribed above for whole antibodies. For example, F(ab)₂ fragments canbe generated by treating antibody with pepsin. The resulting F(ab)₂fragment can be treated to reduce disulfide bridges to produce Fabfragments. The antibody of the present invention is ftirther intended toinclude bispecific and chimeric molecules having an Elf-1 affinityconferred by at least one CDR region of the antibody.

Both monoclonal and polyclonal antibodies (Ab) directed against Elf-1polypeptide or Elf-1 polypeptide variants, and antibody fragments suchas Fab and F(ab)₂, can be used to block the action of Elf-1 and allowthe study of the role of Elf-1 in, for example. embryogenesis and/ortumorogenesis. For example, purified monoclonal Abs can be injecteddirectly into the limb buds of chick or mouse embryos. It isdemonstrated in the examples below that Elf-1 is expressed in the limbbuds of day 10.5 embryos. Thus, the use of anti-Elf-1 Abs during thisdevelopmental stage can allow assessment of the effect of Elf-1 on theformation of limbs in vivo. In a similar approach, hybridomas producinganti-Elf-1 monoclonal Abs, or biodegradable gels in which anti-Elf-1 Absare suspended, can be implanted at a site proximal or within the area atwhich Elf-1 action is intended to be blocked. Experiments of this naturecan aid in deciphering the role of this and other factors that may beinvolved in limb patterning and tissue formation.

Antibodies which specifically bind Elf-1 polypeptide epitopes can alsobe used in immunohistochemical staining of tissue samples in order toevaluate the abundance and pattern of expression of each of the subjectElf-1 polypeptides. Anti-Elf-1 antibodies can be used diagnostically inimmuno-precipitation and immuno-blotting to detect and evaluate Elf-1protein levels in tissue or bodily fluid as part of a clinical testingprocedure. For instance such measurements can be useful in predictivevaluations of the onset or progression of neurological disorders, suchas those marked by denervation-like or disuse-like symptoms. Likewise,the ability to monitor Elf-1 levels in an individual can allowdetermination of the efficacy of a given treatment regimen for anindividual afflicted with such a disorder. The level of Elf-1polypeptides can be measured in bodily fluid, such as in samples ofcerebral spinal fluid, or can be measured in tissue, such as produced bybiopsy. Diagnostic assays using anti-Elf-1 antibodies can include, forexample, immunoassays designed to aid in early diagnosis of aneurodegenerative disorder, particularly ones which are manifest atbirth. Diagnostic assays using anti-Elf-1 polypeptide antibodies canalso include immunloassays designed to aid in early diagnosis andphenotyping of a neoplastic or hyperplastic disorder.

Another application of anti-Elf-1 antibodies of the present invention isin the immunological screening of cDNA libraries constructed inexpression vectors such as λgt11, λgt18-23, λZAP, and λORF8. Messengerlibraries of this type, having coding sequences inserted in the correctreading frame and orientation, can produce fusion proteins. Forinstance. λgt11 will produce fusion proteins whose amino termini consistof β-galactosidase amino acid sequences and whose carboxy terminiconsist of a foreign polypeptide. Antigenic epitopes of an Elf-1 proteincan then be detected with antibodies, as, for example, reactingnitrocellulose filters lifted from infected plates with anti-Elf-1antibodies. Positive phage detected by this assay can then be isolatedfrom the infected plate. Thus, the presence of Elf-1 homologs can bedetected and cloned from other animals, as can alternate isoforms(including splicing variants).

Moreover, the nucleotide sequence determined from the cloning of theElf-1 gene will further allow for the generation of probes and primersdesigned for use in identifying and/or cloning Elf-1 homologs in othercell types, e.g. from other tissues, as well as Elf-1 homologs fromother animals, e.g. humans. For instance, the present invention alsoprovides a probe/primer comprising a substantially purifiedoliigonucleotide, which oligonucleotide comprises a region of nucleotidesequence that hybridizes under stringent conditions to at least 10consecutive nucleotides of sense or anti-sense sequence of SEQ ID No: 1,or naturally occurring mutants thereof. For instance, primers based onthe nucleic acid represented in SEQ ID No. 1 can be used in PCRreactions to clone Elf-1 homologs. Likewise, probes based on the Elf-1sequence of SEQ ID No. 1 can be used to detect Elf-1 transcripts orgenomic sequences. In preferred embodiments, the probe futher comprisesa label group attached thereto and able to be detected, e.g. the labelgroup is selected from the group consisting of radioisotopes,fluorescent compounds, enzymes, and enzyme co-factors. Such probes canalso be used as a part of a diagnostic test kit for identifying cells inwhich Elf-1 is misexpressed, such as by measuring a level of an Elf-1encoding nucleic acid in a sample of cells from a patient, e.g.detecting Elf-1 mRNA levels or determining whether a genomic Elf-1 genehas been mutated or deleted.

To illustrate, nucleotide probes can be generated from the Elf-1 genewhich facilitate histological screening of intact tissue and tissuesamples for the presence of an Elf-1 polypeptide mRNA. Similar to thediagnostic uses of anti-Elf-1 polypeptide antibodies, the use of probesdirected to Elf-1 messages, or to geniomic Elf-1 sequences, can be usedfor both predictive and therapeutic evaluation of allelic mutationswhich might be manifest in, for example, neoplastic or hyperplasticdisorders (e.g. unwanted cell growth) or abnormal differentiation oftissue. Used in conjunction with anti-Elf-1 immunoassays, the nucleotideprobes can help facilitate the determination of the molecular basis fora developmental disorder which may involve some abnormality associatedwith expression (or lack thereof) of an Elf-1 polypeptide. For instance,variation in Elf-1 polypeptide synthesis can be differentiated from amutation in the Elf-1 coding sequence.

Accordingly, the present method provides a method for determining if asubject is at risk for a disorder characterized by unwanted cellproliferation or abherelnt control of differentiation. In preferredembodiments, the subject method can be generally characterized ascomprising detecting, in a tissue sample of the subject (e.g. a humanpatient), the presence or absence of a genetic lesion characterized byat least one of (i) a mutation of a gene encoding an Elf-1 polypeptideor (ii) the mis-expression of an Elf-1 gene. To illustrate, such geneticlesions can be detected by ascertaining the existence of at least one of(i) a deletion of one or more nucleotides from an Elf-1 genye, (ii) anaddition of one or more nucleotides to such an Elf-1 gene, (iii) asubstitution of one or more nucleotides of an Elf-1 gene, (iv) a grosschromosomal rearrangement of an Eff-1 genes, (v) a gross alteration inthe level of a messenger RNA transcript of an Elf-1 gene, (vi) thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of an Elf-1 gene, and (vii) a non-wild type level of an Elf-1polypeptide. In one aspect of the invention there is provided aprobe/primer comprising an oligonucleotide containing a region ofnucleotide sequence which is capable of hybridizing to a sense orantisense sequence of SEQ ID No: 1, or naturally occurring mutantsthereof, or 5′ or 3′ flanking sequences or intronic sequences naturallyassociated with an Elf-1 g,cne. The probe is exposed to nucleic acid ofa tissue sample; and the hybridization of the probe to the samplenucleic acid is detected. In certain embodiments, detection of thelesion comprises utilizing the probe/primer in a polymerase chainreaction (PCR) (see e.g., U.S. Pat. No: 4,683,195 and 4,683,202) or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science, 241:1077-1080: and NaKazawa et al. (1944) PNAS91:360-364) the later of which can be particularly useful for detectingpoint mutations in the Elf-1 gene. Alternatively, immunioassays can beemployed to determine the level of Elf-1 protein, either soluble ormembrane bound.

Also, the use of anti-sense techniques (e.g. microinjection of antisensemolecules, or transfection with plasmids whose transcripts areanti-sense with regard to an Elf-1 mRNA or gene sequence) can be used toinvestigate role of Elf-1 in developmental events, as well as the normalcellular function of Elf-1 in adult tissue. Such techniques can beutilized in cell culture, but can also be used in the creation oftransgenic animals.

Furthermore, by making available purified and recombinant Elf-1polypeptides, the present invention facilitates the development ofassays which can be used to screen for drugs, or for Elf-1 homologs,which are either agonists or antagonists of the normal cellular functionof the subject Elf-1 polypeptides, or of their role in the pathogenesisof cellular proliferation and/or differentiation and disorders relatedthereto. In one embodiment, the assay evaluates the ability of acompound to modulate binding between an Elf-1 polypeptide and an EPHreceptor. A variety of assay formats will suffice and, in light of thepresent inventions, will be comprehended by skilled artisan.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest it, an alteration of binding affinity with receptorproteins. Accordingly, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an EPH receptorpolypeptide which is ordinarily capable of binding an Elf-1 protein. Tothe mixture of the compound and receptor is then added a compositioncontaining a Elf-1 polypeptide. Detection and quantification ofreceptor/Elf-1 complexes provides a means for determining the compound'sefficacy at inhibiting (or potentiating) complex formation between thereceptor protein and the Elf-1 polypeptide. The efficacy of the compoundcan be assessed by generating dose response curves from data obtainedusing various concentrations of the test compound. Moreover, a controlassay can also be performed to provide a baseline for comparison. In thecontrol assay, isolated and purified Elf-1 polypeptide is added to acomposition containing the receptor protein, and the formation ofreceptor/Elf-1 complex is quantitated in the absence of the testcompound.

Complex formation between the Elf-1 polypeptide and an EPH receptor maybe detected by a variety of techniques. For instance, modulation of theformation of complexes can be quantitated using, for example, detectablylabelled proteins such as radiolabelled, fluorescently labelled, orenzymatically labelled Elf-1 polypeptides, by immunoassay, or bychromatographic detection.

Typically, it will be desirable to immobilize either the EPH receptor orthe Elf-1 polypeptide to facilitate separation of receptor/Elf-1complexes from uLncomplexed forms of one of the proteins, as well as toaccomadate automation of the assay. In one embodiment, a fusion proteincan be provided which adds a domain that allows the protein to be boundto a matrix. For example, glutathione-S-transferase/receptor(GST/receptor) fusion proteins can be adsorbed onto glutathionesepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathionederivatized microtitre plates, which are then combined with the Elf-1polypeptide, e.g. an ³⁵S-labeled Elf-1 polypeptide, and the testcompound and incubated under conditions conducive to complex formation,e.g. at physiological conditions for salt and pH, though slightly morestringent conditions may be desired, e.g., at 4° C. in a buffercontaining 0.6M NaCl or a detergent such as 0.1% Triton X-00. Followingincubation, the beads are washed to remove any unbound Elf-1polypeptide, and the matrix bead-bound radiolabel determined directly(e.g. beads placed in scintilant), or in the superntantant after thereceptor/Elf-1 complexes are dissociated. Alternatively, the complexescan dissociated from the bead, separated by SDS-PAGE gel, and the levelof Elf-1 polypeptide found in the bead fraction quantitated from the gelusing standard electrophoretic techniques.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, the EPH receptorprotein can be immobilized utilizing conjugation of biotin anidstreptavidin. For instance, biotinylated receptor molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals. Rockford,Ill.), and immobilized in the wells of strcptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with theEPH receptor but which do not interfere with Elf-1 binding can bederivatized to the wells of the plate, and the receptor trapped in thewells by antibody conjugation. As above, preparations of an Elf-1polypeptide and a test compound are incubated in the receptor-presentingwells of the plate, and the amount of receptor/Elf-1 complex trapped inthe well can be quantitated. Exemplary methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the Elf-1 polypeptide, or which are reactive with thereceptor protein and compete for binding with the Elf-1 polypeptide; aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the Elf-1 polypeptide. In the instance of thelatter, the enzyme can be chemically conjugated or provided as a fusionprotein with the Elf-1 polypeptide. To illustrate, the Elf-1 polypeptidecan be chemically cross-linked or genetically fused with alkalinephosphatase, and the amount of Elf-1 polypeptide trapped in the complexcan be assessed with a chromogenic substrate of the enzyme, e.g.paranitrophenylphosphate. Likewise, a fusion protein comprising theElf-1 polypeptide and glutathione-S-transferase can be provided, andcomplex formation quantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quanatitating one of theproteins trapped in the complex, antibodies against the protein, such asthe anti-Elf-1 antibodies described hercin, can be used. Alternatively,the protein to be detected in the complex can be “epitope tagged” in theform of a fusion protein which includes, in addition to the Elf-1polypeptide or EPH receptor sequence, a second polypeptide for whichantibodies are readily available (e.g. from commercial sources). Forinstance, the GST fusion proteins described above can also be used forquantification of binding using antibodies against the GST moiety. Otheruseful epitope tans include myc-epitopes (e.g., see Lillison et al.(1991) J Biol Chem 266.21150-21157) which includes a 10-residue sequencefrom c-myc, as well as the pFLAG system (International Biotechnologies,Inc.) or the pEZZ-protein A system (Pharamacia, N.J.).

Another aspect of the present invention relates to a method of inducingand/or maintaining a differentiated state. enhancing survival, and/orpromoting proliferation of a cell responsive to an Elf-1 protein, bycontacting the cells with an Elf-1 agonist or an Elf-1 antagonist. Forinstance, it is contemplated by the invention that, in light of thepresent finding of an apparently broad involvement of Elf-1 proteins inthe formation of ordered spatial arrangements of differentiated tissuesin vertebrates, the subjcct method could be used to generate and/ormaintain an array of different vertebrate tissue both in vitro and invivo. The Elf-1 agent can be, as appropriate, any of the preparationsdescribed above, including isolated polypeptidcs, gene therapyconstructs, antisensc molecules, peptidomimctics or agents identified inthe drug assays provided herein.

For example, the present method is applicable to cell culture technique.In vitro neuronal culture systems have proved to be fundamental andindispensable tools for the study of neural development, as well as theidentification of neurotrophic factors such as nerve growth factor(NGF), ciliary trophic factors (CNTF), and brain derived neurotrophicfactor (BDNF). Once a neuronal cell has become terminally-differentiatedit typically will not change to another terminally differentiatedcell-type. However, neuronal cells can nevertheless readily lose theirdifferentiated state. This is commonly observed when they are grown inculture from adult tissue, and when they form a blastema duringregeneration. The present method provides a means for ensuring anadequately restrictive environment in order to maintain neuronal cellsat various stages of differentiation, and can be employed, for instance,in cell cultures designed to test the specific activities of othertrophic factors. In such embodiments of the subject method, the culturedcells can be contacted with an Elf-1 polypeptide, or an agent identifedin the assays described above, in order to induce neuronaldifferentiation (e.g. of a stem cell), or to maintain the integrity of aculture of terminally-differentiated neuronal cells by preventing lossof differentiation. The source of Elf-1 in the culture can be derivedfrom, for example, a purified or semi-purified protein composition addeddirectly to the cell culture media, or alternatively, released from apolymeric device which supports the growth of various neuronal cells andwhich has been doped with a Elf-1 protein. The source of the Elf-1 canalso be a cell that is co-cultured with the intended neuronal cell andwhich produces a recombinant Elf-1. Alternatively, the source can be theneuronal cell itself which as been engineered to produce a recombinantElf-1. In an exemplary embodiment, a naive neuronal cell (e.g. a stemcell) is treated with a Elf-1 agonist in order to induce differentiationof the cells into, for example, sensory neurons or, alternatively,motorneurons. Such neuronal cultures can be used as convenient assaysystems as well as sources of implantable cells for therapeutictreatments. For example, Elf-1 polypeptides may be useful inestablishing and maintaining the olfactory neuron cultures describedU.S. Pat. No. 5,318,907 and the like.

To further illustrate potential uses, it is noted that intracerebralgrafting has emerged as an additional approach to central nervous systemtherapies. For example, one approach to repairing damaged brain tissuesinvolves the transplantation of cells from fetal or neonatal animalsinto the adult brain (Dunnett et al. (1987) J Exp Biol 123:265-289; andFreund et al. (1985) J Neurosci 5:603-616). Fetal neurons from a varietyof brain regions can be successfully incorporated into the adult brain,and such grafts can alleviate behavioral defects. For example, movementdisorder induced by lesions of dopaminergic projections to the basalganglia can be prevented by grafts of embryonic dopaminergic neurons.Complex cognitive functions that are impaired after lesions of theneocortex can also be partially restored by grafts of embryonic corticalcells. Thus, use of the present EPH receptor ligands for maintenance ofneuronal cell cultures can help to provide a source of implantableneuronal tissue. The use of an Elf-1 polypeptide in the culture can beto prevent loss of differentiation, or where fetal tissue is used,especially neuronal stem cells, an Elf-1 polypeptide can be used toinduce differentiation.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells, andothers of which can generate only one type of cell, such as sensoryneurons, and likely represent committed progenitor cells. The role of anElf-1 protein employed in the present method to culture such stem cellscan be to induce differentiation of the uncommitted progenitor andthereby give rise to a committed progenitor cell, or to cause furtherrestriction of the developmental fate of a committed progenitor celltowards becoming a terminally-differentiated neuronal cell, for example,the present method can be used in vitro to induce and/or maintain thedifferentiation of neural crest cells into glial cells, schwann cells,chromaffin cells, cholinergic sympathetic or parasympathetic neurons, aswell as peptidergic and serotonergic neurons. The Elf-1 polypeptide canbe used alone, or can be used in combination with other neurotrophicfactors which act to more particularly enhance a particulardifferentiation fate of the neuronal progenitor cell. In the laterinstance, the Elf-1 polypeptide might be viewed as ensuring that thetreated cell has achieved a particular phenotypic state such that thecell is poised along a certain developmental pathway so as to beproperly induced upon contact with a secondary neurotrophic factor. Insimilar fashion, even relatively undifferentiated stem cells orprimative neuroblasts can be maintained in culture and caused todifferentiate with treatment of Elf-1 polypeptides. Exemplary primativecell cultures comprise cells harvested from the nueral plate or neuraltube of an embryo even before much overt differentiation has occurred.

In addition to the implantation of cells cultured in the presence of afunctional Elf-1 activity, yet another objective of the presentinvention concerns the therapeutic application of an Elf-1 polypeptideor mimetic to enhance survival of neurons and other neulonal cells inboth the central nervous system and the peripheral nervous system. Theability of Elf-1 to regulate neuronal differentiation and survivalduring development of the nervous system and also presumably in theadult state indicates that Elf-1 can be reasonably expected tofacilitate control of adult neurons with regard to maintenance,functional performance, and aging of normal cells; repair andregeneration processes in chemically or mechanically lesioned cells; andprevention of degeneration and premature death which result from loss ofdifferentiation in certain pathological conditions. In light of thisunderstanding, the present invention specifically contemplatesapplications of the subject proteins to the treatment of (preventionand/or reduction of the severity of) neurological conditions derivingfrom: (i) acute subacute, or chronic injury to the nervous system,including traumatic injury, chemical injury, vasal injury and deficits(such as the ischemia resulting from stroke), together withinfectious/inflammatory and tumor-induced injury; (ii) aging of thenervous system including Alzheimer's disease; (iii) chronicneurodegenerative diseases of the nervous system, including Parkinson'sdisease, Huntington's chorea, amylotrophic lateral sclerosis and thelike, as well as spinocerebellar degenerations; (iv) chronicimmunological diseases of the nervous system or affecting the nervoussystem, including multiple sclerosis; and (v) disorders of sensoryneurons as well as degenerative diseases of the retina.

Many neurological disorders are associated with degeneration of discretepopulations of neuronal elements and may be treatable with a therapeuticregimen which includes an Elf-1 polypeptide (or equivalent thereof). Forexample, Alzheimer's disease is associated with deficits in severalneurotransmitter systems, both those that project to the neocortex andthose that reside with the cortex. For instance, the nucleus basalis inpatients with Alzheimer's disease were observed to have a profound (75%)loss of neurons compared to age-matched controls. Although Alzheimer'sdisease is by far the most common form of dementia, several otherdisorders can produce dementia. Several of these are degenerativediseases characterized by the death of neurons in various parts of thecentral nervous system, especially the cerebral cortex. However, someforms of dementia are associated with degeneration of the thalmus or thewhite matter underlying the cerebral cortex. Here, the cognitivedysfunction results from the isolation of cortical areas by thedegeneration of efferents and afferents. Huntington's disease involvesthe degeneration of intrastraital and cortical cholinergic neurons andGABAergic neurons. Pick's disease is a severe neuronal degeneration inthe neocortex of the frontal and anterior temporal lobes, sometimesaccompanied by death of neurons in the striatum. Treatment of patientssuffering from such degenerative conditions can include the applicationof Elf-1 polypeptides, or agents which mimic their effects, in order tomanipulate, for example, the de-differentiation and apoptosis of neuronswhich give rise to loss of neurons. In preferred embodiments, a sourceof an Elf-1 agent is stereotactically provided within or proximate thearea of degeneration.

In addition to degenerative-induced dementias, a pharmaceuticalpreparation of an Elf-1 homolog can be applied opportunely in thetreatment of neurodegenerative disorders which have manifestations oftremors and involuntary movements. Parkinson's disease, for example,primarily affects subcortical structures and is characterized bydegeneration of the nigrostriatal pathway, raphe nuclei, locus cereleus,and the motor nucleus of vagus. Ballism is typically associated withdamage to the subthalmic nucleus, often due to acute vascular accident.Also included are neurogenic and myopathic diseases which ultimatelyaffect the somatic division of the peripheral nervous system and aremanifest as neuromuscular disorders. Examples include chronic atrophiessuch as amyotrophic lateral sclerosis, Guillain-Barre syndrome andchronic peripheral neuropathy, as well as other diseases which can bemanifest as prog,rcssive bulbar palsies or spinal muscular atrophies.The present method is ammenable to the treatment of disorders of thecerebellum which result in hypotonia or ataxia, such as those lesions inthe cerebellum which produce disorders in the limbs ipsilateral to thelesion. For instance, a preparation of an Elf-1 homolog can be used totreat a restricted form of cerebellar corical degeneration involving theanterior lobes (vermis and leg areas) such as is common in alcoholicpatients.

In an yet another embodiment, the subject method is used to treatamyotrophic lateral sclerosis. ALS is a name given to a complex ofdisorders that comprise upper and lower motor neurons. Patients maypresent with progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, or a combination of these conditions.The major pathological adnomality is characterized by a selective andprogressive degeneration of the lower motor neurons in the spinal cordand the upper motor neurons in the cerebral cortex. The therapeuticapplication of an Elf-1 therapeutic agent, such as a soluble form of theprotein represented in SEQ ID No: 2 or a peptidomimetic thereof, can beused alone or in conjunction with other neurotrophic factors such asCNTF, BDNF, or NGF to prevent and/or reverse motor neuron degenerationin ALS patients

The Elf-1 polypeptides of the present invention can also be used in thetreatment of autonomic disorders of the peripheral nervous system, whichinclude disorders affecting the innervation of smooth muscle andendocrine tissue (such as glandular tissue). For instance, Elf-1compositions may be useful to treat tachycardia or atrial cardiacarrythmias which may arise from a degenerative condition of the nervesinnervating the striated muscle of the heart.

Furthermore, a potential role of Elf-1 which is apparent from theappended examples, namely the data respecting Elf-1 and its cognatereceptors in retinal tissue and in the limb bud, concerns the role ofElf-1 in development and maintenance of dendritic processes of axonalneurons. In particular, as set forth in the examples below, Elf-1 isexpressed in the midbrain. This region is where several different senseorgans project and form a topographic representation of the outsideworld. For example, the retinal ganglion cells project onto the tectum(which is a part of the midbrain). The in silit data described belowsuggests that the sek and mek4 (a hek-related receptor) are expressed inthe developing optic cup and possibly optic vesicle. Moreove, it hasbeen reported elsewhere that cek4 and cek8 are expressed in retinaltissue (Sajjadi et al. (1993) Oncogene 8:1807-1813). In light of theseexpression patterns, it is submitted herein that Elf-1 may be along-sought factor that controls projection of neurons from the senseorgans onto the topographic maps of the brain (for review, see Holt etal. (1993) J Neurobiol 24:1400-1422). In addition to providing guidancefor the axonal projections, it is quite plausible that Elf-1 couldpromote the differentiation and/or maintenance of the innervating cellsto their axonal processes.

Accordingly, compositions comprising Elf-1 homologs or other Elf-1agents described herein may be employed to support or alternatively,antagonize the survival and reprojection of several types of central andperipheral ganglionic neurons, sympathetic and sensory neurons, as wellas motor neurons. In particular, such therapeutic compositions may beuseful in treatments designed to rescue, for example, retinal ganglia,inner ear and accoustical nerves, and motorneurons, from lesion-induceddeath as well as guiding reprojection of these neurons after suchdamage. Such diseases and conditions include but are not limited to CNStrauma, infaretion, infection (such as viral infection withvaricella-zoster), metabolic disease, nutritional deficiency, toxicagents (such as cisplatin treatment). Moreover, certain of the Elf-1agents (probably antagonistic forms) may be useful in the selectiveablation of sensory neurons, for example in the treatment of chronicpain syndromes.

Elf-1 can be used in nerve prostheses for the repair of central andperipheral nerve damage. In particular, where a crushed or severed axonis entubulated by use of a prosthetic device, Elf-1 polypeptides can beadded to the prosthetic device to increase the rate of growth andregeneration of the dendritic processes. Exemplary nerve guidancechannels are described in U.S. Pat. Nos. 5,092,871 and 4,955,892.Accordingly, a severed axonal process can be directed toward the nerveending from which it was severed by a prosthesis nerve guide whichcontains, e.g. a semi-solid formulation containing an Elf-1 polypeptideor mimetic, or which is derivatized along the inner walls with an Elf-1protein.

Moreover, compositions of Elf-1 polypeptides may be useful in thetreatment of retinal degeneration. e.g., to enhance survival andprojection of retinal ganglion cells, as well as to possibly rescueretinal photoreceptor cells. Such therapeutic intervention could includeadministration of an Elf-1 composition alone or in conjuntion with othersurvival/growth factors. Corrective gene therapy with an Elf-1 geneconstruct described above may also be performed. Suitable implants forintraoccular delivery of Elf-1 preparations can be found in. forexample, Park et al. (1993) Int Rev Cytol 146:49-74; Ben-Nun et al.(1989) Aust N Z J Ophthalmol 17:185-190; and U.S. Pat. No. 5,273,530.

In yet another embodiment, the subject Elf-1 polypeptides can be used inthe treatment of ncoplastic or hyperplastic transformations, particularyof the central nervous system and lymphatic system. For instance,certain Elf-1 homologs are likely to be capable of inducingdifferentiation of transformed neuronal cells to become post-mitotic orpossibly apoptotic. Treatment with other Elf-1 homologs may involvedisruption of autocrine loops, such as TGF-β or PDGF autostimulatoryloops. believed to be involved in the neoplastic transformation ofseveral neuronal tumors. Elf-1 homologs may, therefore, be of use in thetreatment of, for example, malignant gliomas, medulloblastomas,neuroectodermal tumors, and ependymonas.

Likewise, the EPH receptor hek, which is the human ortholog of mek4, hasbeen shown to be expressed on the surface of non-neuronal tumor cells,and its hyperexpression in tumor cells has been suggested to play a rolein tumor induction. Accordingly, antagonist of Elf-1 (includingantisense and gene therapy contructs) may be useful in the treatment of,for example, hematopoietic tumors and other tumors which express EPHreceptors which bind EIf-1.

Yet another aspect of the present invention concerns the application ofthe discovery that Elf-1 proteins are presumably induction signalsinvolved in other vertebrate organogenic pathways in addition toneuronal differentiation as described above, having potential roles inother ectodermal patterning, as well as both mesodermal and endodermaldifferentiation processes. Thus, it is contemplated by the inventionthat compositions comprising Elf-1 proteins can also be utilized forboth cell culture and therapeutic methods involving, generation andmaintenance of non-neuronal tissue, such as in controlling thedevelopment and maintenance of tissue from the digestive tract, liver,lungs, and other organs which derive from the primitive gut, as well asdorsal mesoderm-derived structures including muscular-skeletal tissuesand connective tissue of the skin, intermediate mesoderm-derivedstructures, such as the kidney and other renal and urogenital tissues;and head mesenchylllal and neural crest-derived tissue, such as cephalicconnective tissue and skull and branchial cartilage, occular tissue,muscle and cardiac tissue. This should not be construed as acomprehensive list, and other tissues which may be affected by Elf-1polypeptides are envisaged.

For example, Elf-1 polypeptides can be employed in the development andmaintenance of an artificial liver which can have multiple metabolicfunctions of a normal liver. In an exemplary embodiment. Elf-1 agonistsand/or antagonists can be used, optionally with other growth anddifferentiation factors, to cause differentiation of digestive tube stemcells to form hepatocyte cultures which can be used to populateextracellular matrices, or which can be encapsulated in biocompatiblepolymers, to form both implantable and extracorporeal artificial livers.Likewise, therapeutic compositions of certain of the subject Elf-1polypetides can be utilized in conjunction with transplantation of suchartificial livers, as well as embryonic liver structures, to promoteintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue, as wellas to regulate such organs after physical, chemical or pathologicalinsult.

Similarly, therapeutic compositions containing Elf-1 proteins can beused to promote regeneration of lung tissue in the treatment ofemphysema and other degenerative conditions of the lung. For example.Elf-1 compositions may useful in the treatment of degenerative disordersof lung tissue caused by, for instance, toxic injuries, as well asinflammatory and degenerative processes induced by viral infections.Tissue degeneration of the lung, and hence the therapeutic target for anElf-1 composition, includes degenerative changes affecting theendothelial and epithelial cells, basal membrane, and mesenchymal andmatrix structures.

In still another embodiment of the present invention, compositionscomprising Elf-1 polypeptides of the present invention can be used inthe in vitro generation of skeletal tissue such as from skeletogenicstem cells, as well as the in vivo treatment of skeletal tissuedeficiencies. The present invention particularly contemplates the use ofElf-1 homologs which maintain a skeletal homeotic activity, such as anability to induce chondrogenesis and/or osteogenesis. By “skeletaltissue deficiency”, it is meant a deficiency in bone or other skeletalconnective tissue at any site where it is desired to restore the bone orconnective tissue, no matter how the deficiency orioinated, e.g. whetheras a result of surgical intervention, removal of tumor, ulceration,implant, fracture, or other traumatic or degenerative conditions.

For instance, the present invention makes available effectivetherapeutic methods and compositions for restoring cartilage function toa connective tissue. Such Elf-1 compositions are useful in, for example,the repair of defects or lesions in cartilage tissue which is the resultof degenerative wear such as that which results in arthritis, as well asother mechanical derangements which may be caused by trauma to thetissue, such as a displacement of torn meniscus tissue, meniscectomy, alaxation of a joint by a torn ligament, malignment of joints, bonefracture, or by hereditary disease. The present reparative method isalso useful for remodeling cartilage matrix, such as in plastic orreconstructive surgery, as well as periodontal surgery. The presentmethod may also be applied to improving a previous reparative procedure,for example, following surgical repair of a meniscus, ligament, orcartilage. Furthermore, it may prevent the onset or exacerbation ofdegenerative disease if applied early enough after trauma. In oneembodiment of the present invention, the subject method comprisestreating the afflicted connective tissue with a therapeuticallysufficient amount of an Elf-1 polypeptide to generate a cartilage repairresponse in the connective tissue by stimulating the differentiationand/or proliferation of chondrocytes embedded in the tissue.

The present invention further contemplates the use of the subject methodin the field of cartilage transplantation and prosthetic devicetherapies. For instance, the action of chondrogensis in the implantedtissue, as provided by the subject method, and the mechanical forces onthe actively remodeling tissue can synergize to produce an improvedimplant more suitable for the new function to which it is to be put.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. The need for improved treatment has motivated researehaimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 71B:74; Vacanti et al. (1991) Plast Reconstr Surg88:753; von Schroeder et al. (1991) J. Biomed Mater Res 25:329; Freed etal. (1993) J Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scafiolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers which degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own ear or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

In one embodiment of the subject method, the implants are contacted withan Elf-1 polypeptide during the culturing process, such as an Elf-1agonist, in order to induce and/or maintain differentiated chondrocytesin the culture so as to further stimulate cartilage matrix productionwithin the implant. In such a manner, the cultured cells can be causedto maintain a phenotype typical or a chondrogenic cell (i.e.hypertrophic), and hence continue the population of the matrix andproduction of cartilage tissue.

In still further embodiments, the subject method can be employed for thegeneration of bone (osteogenesis) at a site in the animal where suchskeletal tissue is deficient. Thus, preparations comprising Elf-1polypeptides can be employed, for example, to induce endochondralossification, at least so far as to facilitate the formation ofcartilaginous tissue precursors to form the “model” for ossification.Therapeutic compositions of Elf-1 polypeptides can be supplemented, ifrequired, with other osteoinductive factors, such as bone morphogenicproteins (and other TGF-β factors).

In yet another embodiment of the present invention, an Elf-1 antagonistcan be used to inhibit spermatogenesis. Thus, in light of the pastfinding that the mek4 receptors are localized in testicular tissue, itis possible that Elf-1 proteins are involved in the differentiationand/or proliferation and maintenance of testicular germ cells.Accordingly, Elf-1 antagonist can be utilized to block the action of anaturally-occuring Elf-1 protein. In a preferred embodiment, tie Elf-1antagonist inhibits the biological activity of an authentic Elf-1homolog in spermatogenesis by competitvelv binding EPH receptors, suchas mek4, in the testis. In similar fashion, Elf-1 agonists andantagonists are potentially useful for modulating normal ovarianfunction.

The Elf-1 polypeptides of the present invention, or pharmaceuticallyacceptable salts thereof, may be conveniently formulated foradministration with a biologically acceptable medium. such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Theoptimum concentration of the active ingredient(s) in the chosen mediumcan be determined emperically, according to procedures well known tomedicinal chemists. As used herein, “biologically acceptable medium”includes any and all solvents, dispersion media, and the like which maybe appropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activivity of theElf-1 polypeptide, its use in the pharamceutical preparation of theinvention is contemplated. Suitable vehicles and their formulationinclusive of other proteins are described, for example, in the bookRemingion's Pharmaceutical Sciences (Remington's PharmaceuticalSciences. Mack Publishing Company, Easton, Pa., USA 1985). Thesevehicles include injectable “deposit formulations”. Based on the above,such pharmaceutical formulations include, although not exclusively,solutions or freeze-dried powders of an Elf-1 polypeptide in associationwith one or more pharmaceutically acceptable vehicles or diluents, andcontained in buffered media at a suitable pH and isosmotic withphysiological fluids. For illustrative purposes only and without beinglimited by the same, possible compositions or formulations which may beprepared in the form of solutions for the treatment of nervous sytemdisorders with an Elf-1 polypeptide are given in U.S. Pat. No.5,218,094. In the case of freeze-dried preparations, supportingexcipients such as, but not exclusively, mannitol or glycine may be usedand appropriate buffered solutions of the desired volume will beprovided so as to obtain adequate isotonic buffered solutions of thedesired pH. Similar solutions may also be used for the pharmaceuticalcompositions of Elf-1 polypeptides in isotonic solutions of the desiredvolume and include, but not exclusively, the use of buffered salinesolutions with phosphate or citrate at suitable concentrations so as toobtain at all times isotonic pharmaceutical preparations of the desiredpH, (for example, neutral pH).

Methods of introduction of exogenous Elf-1 polypeptides at the site oftreatment include, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, oral, and intranasal. Inaddition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injectioni.Intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir.

Methods of introduction may also be provided by rechargable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of an Elf-1 at a particular targetsite. Such embodiments of the present invention can be used for thedelivery of an exogenously purified Elf-1 polypeptides, which has beenincorporated in the polymeric device, or for the delivery of Elf-1polypeptides produced by a cell encapsulated in the polymeric device.The generation of such implants is generally known in the art. See, forexample, Concise Encylopedia of Medical & Dental Materials, ed. by DavidWilliams (MIT Press: Cambridge, Mass., 1990); the Sabel et al. U.S. Pat.No. 4,883,666; Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer etal. U.S. Pat. No. 5,106,627; Lim U.S. Pat. No. 4,391,909; and SeftonU.S. Pat. No. 4,353,888.

In yet another embodiment of the present invention, the pharmaceuticalElf-1 polypeptide can be administered as part of a combinatorial therapywith other agents. For example, the combinatorial therapy can include anElf-1 protein with at least one trophic factor. Exemplary trophicfactors include nerve growth factor, cilliary neurotrophic growthfactor, schwanoma-derived growth factor, glial growth factor,stiatal-derived neuronotrophic factor, platelet-derived growth factor,and scatter factor (HGF-SF).

Another aspect of the invention features transgenic non-human animalswhich express a heterologous Elf-1 gene of the present invention, orwhich have had one or more genomic Elf-1 gene(s) disrupted in at leastone of the tissue or cell-types of the animal. Accordingly, theinvention features an animal model for developmental diseases, whichanimal has an Elf-1 allele which is mis-expressed. For example, a mousecan be bred which has one or more Elf-1 alleles deleted or otherwiserendered inactive. Such a mouse model can then be used to studydisorders arising from mis-expressed Elf-1 genes.

Another aspect of the present invention concerns transgenic animalswhich are comprised of cells (of that animal) which contain a transgeneof the present invention and which preferably (though optionally)express an exogenous Elf-1 protein in one or more cells in the animal.The Elf-1 transgene can encode the wild-type form of the protein, or canencode homologs thereof, including both agonists and antagonists, aswell as antisense constructs. In preferred embodiments, the expressionof the transgene is restricted to specific subsets of cells, tissues ordevelopmental stages utilizing, for example, cis-acting sequences thatcontrol expression in the desired pattern. In the present invention,such mosiac expression of the subject polypeptide can be essential formany forms of lineage analysis and can additionally provide a means toassess the effects of, for example lack of Elf-1 expression which mightgrossly alter development in small patches of tissue within an otherwisenormal embryo. Toward this and, tissue-specific regulatory sequences andconditional regulatory sequences can be used to control expression ofthe transgene in certain spatial patterns. Moreover, temporal patternsof expression can be provided by, for example, conditional recombinationsystems or prokaryotic transcriptional regulatory sequences.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination a target sequence. As used herein, the phrase“target sequence” refers to a nucleotide sequence that is geneticallyrecombined by a recombinase. The target sequence is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinlasecatalyzed recombination events can be designed such that recombinationof the target sequence results in either the activation or repression ofexpression of the subject Elf-1 polypeptide. For example, excision of atarget sequence which interferes with the expression of a recombinentElf-1 gene, such as one which encodes an antagonistic homolog, can bedesigned to activate expression of that gene. This interference withexpression of the protein can result from a variety of mechanisms, suchas spatial separation of the Elf-1 gene from the promoter element or aninternal stop codon. Moreover, the transgene can be made wherein thecoding sequence of the gene is flanked recombinase recognition sequencesand is initially transfected into cells in a 3′ to 5′ or ientation withrespect to the promoter element. In such an instance, inversion of thetarget sequence will reorient the subject gene by placing the 5′ end ofthe coding sequence in an orientation with respect to the promoterelement which allow for promoter driven transcriptional activation.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al.(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355, PCTpublication WO 92/15694) can be used to generate in vivo site-specificgenetic recombination systems. Cre recombinase catalyzes thesite-specific recombination of an intervening target sequence locatedbetween loxP sequences. loxP sequences are 34 base pair nucleotidcrepeat sequences to which the Cre recombinase binds and are required forCre recombinase mediated genetic recombination. The orientation of loxPsequences determines whether the intervening target sequence is excisedor inverted when Cre recombinase is present (Abremski et al. (1984) J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of the receombinant Elf-1 protein can beregulated via control of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of arecombinant Elf-1 protein requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and arecombinant Elf-1 gene can be provided through the construction of“double” transgenic animals. A convenient method for providing suchanimals is to mate two transgenic animals each containing a transgene,e.g. an Elf-1 gene and recombinase gene.

One advantage derived from initially constructing transgenic animalscontaining a Elf-1 transgene in a recombinase-mediated expressibleformat, particularly derives from the likelihood that the subjectprotein will be deleterious upon expression in the transgenic animal. Insuch an instance, a founder population, in which the subject transgeneis silent in all tissues, can be propagated and maintained. Individualsof this founder population can be crossed with animals expressing therecombinase in, for example, one or more tissues. Thus, the creation ofa founder population in which, for example, an antagonistic Elf-1transgene is silent will allow the study of progeney from that founderin which diruption of Elf-1 mediated induction in a particular tissue orat developmental stages would result in, for example, a lethalphenotype.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the transgene. Exemplarypromoters and the corresponding trans-activating prokaryotic proteinsare given in U.S. Pat. No. 4,833,080. Moreover, expression of theconditional transgenes can be induced by gene therapy-like methodswherein a gene encoding the trans-activating protein, e.g. a recombinaseor a prokaryotic protein, is delivered to the tissue and caused to beexpressed, such as in a cell-type specific manner. By this method, theElf-1 transgene could remain silent into adulthood until “turned on” bythe introduction of the trans-activator.

In an exemplary embodiment, the “transgienic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2 pl of DNA solution. The useof zygotes as a target for gene transfer has a major advantage in thatin most cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). Asa consequence, all cells of the transgenic non-human animal will carrythe incorporated transgene. This will in general also be reflected inthe efficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. Microinjectioniof zygotes is the preferred method for incorporating transgenes inpracticing the invention.

Retroviral infection can also be used to introduce transgene into anonhuman animal. The developing non-human embryo can be cultured inivitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jacnich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector systcm used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgyenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgienes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) PNAS 83: 9065-9069: and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal, The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

Methods of making knock-out or disruption transgenic animals are alsogenerally known. See, for example, Manipuiating the Mouse Embryo, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).Recombinase dependent knockouts can also be generated, e.g. byhomologous recombination to insert rccombinase target sequences flankingportions of an endogenous Elf-1 gene, such that tissue specific and/ortemporal control of inactivation of an Elf-1 allele can be controlled asabove.

Yet another aspect of the invention relates to the novel in situ assayfor detecting receptors or their ligands in tissue samples and whileorganisms. In general, the RAP-in situ assay (for Receptor AffinityProbe) of the present invention comprises (i) providing a hybridmolecule (the affinity probe) including a receptor, or a receptorligand, covalently bonded to an enzymatically active tag, preferably forwhich chromogenic substrates exist, (ii) contacting the tissue ororganism with the affinity probe to form complexes between the probe anda cognate receptor or ligand in the sample, removing unbound probe, and(iii) detecting the affinity complex using a chromogenic substrate forthe enzymatic acitivity associated with the affinity probe. In preferredembodiments, an alkaline phosphatase provides a tag that binds tocommercially available antibodies, allowing co-immunoprecipitationprocedures. More significantly, however, it has an intrinsic enzymeactivity that can be traced quantitatively by simple chromogenic assays,without purification, radioactive labeling, or the use of secondaryreagents. We find that detection usingi the enzyme activity of AP fusionproteins provides a sensitivity at least comparable to other approaches,such as the use of purified and ¹²⁵I labeled reagents.

Other enzymes which can be used in place of alkaline phosphatase includehorseradish peroxidase, β-galactosidase, malate dehydrogenase, yeastalcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, asparaoinase, glucose oxidase, and urease. Otherenzyme labels which are readily detectable by addition of acorresponding chromo(genic substrate are known in the art. See also,Flanagan and Leder PCT Publication WO92/06220. The enzyme label can becovalcntly attached to the receptor or receptor ligand by chemicalcross-linking agents known in the art, such as succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);N-succinimidyl(4-iodoacetyl) aminobenzoate (SMPB), succinimidyl4-(p-maleimidophenyl) butyrate (SMPB), or1-ethyl-3-(3-dimethylamiinioproply) carbodiiumide hydrochloride (EDC).Alternatively, recombinant fusion proteins can be generated as describedin the examples below.

The present method, unlike the prior art methods which had only beencarried out on dispersed cell cultures, provides a means for probingnon-dispersed and wholemounlt tissue and animal samples. The method canbe used to detect patterns of expression for particular receptors andtheir ligands, for measuring the affinity of reccptor/ligandinteractions in tissue samples, as well as for generating drug screeningassays in tissue samples. Moreover, the affinity probe can also be usedin diagnostic screening to determine whether a receptor, e.g. an EPHreceptor, or its ligand, e.g. Elf-1 or B61 or LERK-2 protein, aremisexpressed.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the followinig examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

We have previously reported the use of a soluble receptor-AP fusionprotein to identify and characterize the ligand of the c-kit receptor(Flanagan and Leder, (1990) Cell 63: 185-194; Flanagan et al. (1991)Cell 64:1025-1035). Here we extend this approach and show that mek4-APand sek-AP soluble receptor fusions can be used to localize sites ofligand binding directly in the mouse embryo, in a procedure we termRAP-in situ (for Recptor Affinity Probe). Using this spatialinformation, the imek4-AP and sek-AP reagents were then employed toidentify a ligand clone in an expression library of cDNA from regions ofthe embryo found to express very high levels of ligand. The entireprocess of identifying and cloning a ligand by this procedure requiresno purification or labeling of protein reagents, and can be rapid,simple and inexpensive.

A novel EPH receptor lignad, “Elf-1” was identified by this method, andwe show that it is a ligand that can bind to both mek4-AP and sek-AP.Elf-1 is an unusual tyrosinc kinase ligand in being linked to themembrane through a phosphatidylinositol linkage, a feature that may helpto understand some of the unique properties of the EPH family receptors.The sequence of Elf-1 shares some homology to two other EPH receptorligands, B61 (Bartley et al. (1994) Nature 368:558-561; and Holzman etal. (1990) Mol Cell Biol 10:5830-5838) and LERK-2 (Beckmann et al.(1994) EMBO J13:3757-3762; PCT Publication WO95/1 1384).

The developmental expression of Elf-1 and its receptors mek4 and sek wascompared, revealing complementary domains of expression that delineatepotential roles for these molecules in patterning several developmentalfields of the vertebrate embryo. The identification of Elf-1 has allowedus to initiate studies into its developmental function. RNA in situhybridization indicated Elf-1 is expressed by day 8.5 of mousedevelopment, soon after gastrulation. Earlier stages have not yet beenexamined, but will be of interest, particularly as an apparent homologof the sek receptor was reported to be expressed in the zebrafish fromthe onset of gastrulation (Xu et al. (1994) Development 120:287-299). Wefind that Elf-1 and its receptors mek4 and sek are expressed together inseveral regions of the mouse embryo, including the neural tube, somites,limb buds, retina, and branchial arehes. In each of these regions theexpression of ligand and receptors is strikingly complementary, implyingroles for their interaction.

In the developing neural folds and neural tube the expression of Elf-1,sek and mek4 in complementary patterns in the forebrain) midbrain andhindbrain supports roles in early patterning. In the hindbrain,expression of sek (Nieto et al. (1992) Development 116:11 37-1150) aswell as Elf-1 and mek4 appears to be correlated with the boundaries ofthe rhombomeres. These segmental structures, visible as morphologicalbulges, are known to act as cell lineage compartments as well as domainsfor the expression and action of control molecules such as the Hox genes(Guthrie and Lumsden, (1991) Development 112:221-229). The earlyexpression of Elf-1, mek4 and sek in these structures of the hindbrainsuggests functions in the establishment or compartmentalization of thesesegmental structures, or in the activation of the specific molecularprogram of individual rhombomeres. Early expression of Elf-1, sek andmek4 in the somites similarly suggests roles in the establishment orsubsequent fate specification of these metameric structures. It alsoimplies the possibility of parallel molecular mechanisms operating inthe rhombomeres and somites, the two major segmental structures of thevertebrate embryo.

The expression patterns in the limb bud are also intriguing. In eachlimb bud, Elf-1 RNA appears to be expressed in a diffuse centralpattern, while sek is expressed in a defined band at the distal end, andmek4 is expressed in a posterior proximal region. It is interesting thatthis expression shows some correlation with the structures at the distaltip and posterior margin that are known known to play key roles insetting up the proxino-distal and anterior-posterior axes of thisdevelopmental field (Tabin, (1991) Cell 66:199-217). A similarlycomplemetary pattern of ligand and receptor expression is seen in thebranchial arehes. Moreover, the recent observation of mirror imageduplications following Hoxa-2 deletion suggests that each branchial archmay be a morphogenetic field in some ways analogous to a limb bud(Gendron-Maguire et al. (1993) Cell 75:1317-31; Rijli et al. (1993) Cell75:1333-1349). Patterning in these two areas could therefore involvesimilar molecular mechanisms. Accordingly, our observations set forthbelow delineate potential functions for Elf-1 in several important areasof the vertebrate embryo.

Finally, Elf-1 is expressed in the midbrain. This region is whereseveral different sense organs project and form a topographicrepresentation of the outside world. For example, the retinal ganglioncells project onto the tectum (which is a part of the midbrain). The insitu data described below suggests that the sek and mek4 (a hek-relatedreceptor) are expressed in the developing optic cup and possibly opticvesicle. Moreover, it has been repotted elsewhere that cek4 and cek8 areexpressed in retinal tissue (Sajjadi et al. (1993) Oncogene8:1807-1813). In light of these expression patterns, it is submittedherein that Elf-1 may be a long-sought factor that controls projectionof neurons from the sense organs onto the topographic maps of the brain(for review, see Holt et al. (1993) J Neurobiol 24:1400-1422). Inaddition to providing guidance for the axonal projections, it is quiteplausible that Elf-1 could promote the differentiation and/ormaintenance of the innervating cells to their axonal processes.

The need to generate a complex three-dimensional pattern in developmentimplies that key cellular communication molecules must be able totransmit accurate spatial information. This may explain why, althoughthe first growth factors were identified as soluble molecules, it isincreasingly becoming apparent that many, if not most, polypeptidegrowth factors can exist in forms that are not freely diffusible(Jessell and Melton, (1992) Cell 68:257-270; Massague and Pandiella,(1993) Annu. Rev. Biochem. 62:515-541).

For several ligands of tyrosine kinases, this anchorage is mediated bythe presence of a C-terminal transmembrane domain. In the case of thekit ligand/steel factor, genetic evidence indicates that the presence ofthis transmembrane domain is essential for the molecule to fulfil itsnormal developmental function (Flanagan et al., supra; Brannan et al.,(1991) PNAS 88:4671-4674). The precise biological roles of transmembraneanchorage are not clear, but may include tight localization of ligandactivity, and may also be related to the ability of these ligands tomediate cell-cell adhesion and to promote cell migration. Still othertyrosine kinase ligands are anchored by interactions with proteoglycansor other molecules in the extracellular or pericellular matrix. Theseinteractions not only localize the ligands, but can also have majoreffects on their biological activity (Bernfield et al., (1992) Annu.Rev. Cell Biol. 8:365-393; and Jessell and Melton, supra).

Like many of these other ligands for receptor tyrosine kinases, Elf-1expressed in transfected cells is presented at the cell surface.However, the mechanism of this anchorage is unusual in that itapparently involves a linkage to the membrane via a C-terminalphosphatidylinositol glycan tail. This mode of anchorage exists both incell lines, where cell surface ligand was released by PI-PLC treatment,and also in embryos, where all reactivity in RAP-in situ experimentswith imek4-AP and sek-AP was removed by pretreatment with PI-PLC. LikeElf-1, the related polypeptide B61 also shows cell surface anchoragethat is sensitive to PI-PLC (Holzman et al., supra; Bartley et al.,supra). These findin(gs suggest that phosphatidylinositol linkage mayemerge as a general feature of the EPH receptor ligand family.

The presence of a phosphatidylinositol linkage on Elf-1 and B61 adds anovel dimension to the anchorage mechanisms of the ligands for receptortyrosine kinases. One proposed function of this linkage is to ensurecell-cell interactions. For example, the establishment andcornpartmentalization of the rhombomeres clearly implies the existenceof segment-specific cell-cell interactions. Spatially restrictedcellular interactions such as these could well be mediated by theinteraction of EPH family receptors with liganlds such as Elf-1 anchoredin the membranes of adjacent cells. An immunohistochemical study of thenuk receptor is also intriguing in this regard (Henkemeyer et al.,(1994) Oncogene 9:1001-1014). Expression was detected in the earlynervous system at sites of cell-cell contact, often involving migratingneuronal cells, and in the initial axon outgrowths of the nervoussystem. These observations suggest functions in the guidance of neuronalmigration and in the pathfinding and/or fasciculation of the earliestaxons. The presentation of Elf-1 or other EPH receptor ligands in a cellsurface form with a phosphatidylinositol linkage could play an importantpart in determining the spatial specificity of these unique cell-cellinteractions that play a critical role in early stages of patterning thedeveloping vertebrate embryo.

Example 1 Localization of Ligand(s) for mek4 and sek by RAP iin situ

To seareh for ligands for the EPH family members mek4 and sek. cDNAsencoding the receptor extracellular domains were inserted into thevector APtag-1 (Flanagan and Leder, supra). The resulting constructsencode the receptor extracellular domain, presumed to bind extracellularligand(s), fused to placental alkaline phosphatase (FIG. 1A). Thealkaline phosphatase provides a tag that binds to commercially availableantibodies, allowing co-immunoprecipitation procedures. Moresignificantly, it has an intrinsic enzyme activity that can be tracedquantitatively by simple chromogenic assays, without purification,radioactive labeling, or the use of secondary reagents. We find thatdetection using the enzyme activity of AP fusion proteins provides asensitivity at least comparable to other approaches, such as the use ofpurified and ¹²⁵I labeled reagcnts.

FIG. 1B shows that mek4-AP and sek-AP are secreted proteins, and eachwas produced as a single major polypeptide with the expected apparentmolecular weight of approximately 150 kD. The fusion proteins hadalkaline phosphatase enzyme activity, with a specific activity similarto that reported previously (Flanagan and Leder, supra). Individualclones of transfected cells selected for secretion of high alkalinephosphatase activity produced approximately 5 μg/ml of fusion protein inthe supernatant. For all subsequent experiments described here thesupernatants could be used as a source of the soluble receptor reagentwithout purification.

As the mek4 and sek receptors are known to be expressed at high levelsin embryonic development, we decided to test whether the mek4-AP andsek-AP reagents could be used to detect their ligand(s) directly inmouse embryos. Whole embryos were treated with receptor-AP fusionprotein, washed, and then tested for bound fusion protein using standardhistochemical stains for AP activity. Briefly, whole embryos at day 9.5of development were treated with supernatants containing receptor fusionproteins, or unfused AP as a control, then were washed, fixed andstained for bound AP activity. The thin roof of the fourth ventricle inthe hindbrain was punctured to allow exchange of reagents with the lumenof the neural tube, resulting in a slight distortion of the embryos inthis region. A characteristic pattern of staining was detected wheneither mek4-AP or sek-AP was used to test whole-mount preparations ofmouse embryos. Areas that appeared to show specific staining include thefollowing: midbrain and anterior hindbrain, branchial arches, dorsalface of the spinal cord, somites, and limb buds. Similar staining wasnot seen in controls using unfused AP or when other receptor-AP fusionproteins were used. The patterns seen with mek4-AP and sek-AP weresimilar to one another, with the strongest reactivity in both cases seenin the region of the presumptive midbrain and anterior hindbrain.Reactivity was also seen in other areas, including the region of thesomites, the branchial arches, a stripe down the dorsal face of theneural tube posterior to the hindbrain, limb buds, and retina. Strong,specific staining was also seen in embryos at day 8.5 and 10.5 ofdevelopment. The exact tissues and cell types showing reactivity havenot yet been characterized in detail. In addition, it should be notedthat the conditions used for these experiments were selected to minimizebackground staining and the possibility of losing or denaturing theligand. The short incubation times and the absence of permeabilizingagents may favor stainingt of structures near the surface of the embryo.Also, as the embryos were not fixed prior to treatment with thereceptor-AP reagents, the protocol used here may not be suitable fordetecting freely diffusible ligands. However, in other experiments wefind that the RAP-in situ procedure can be modified to use fixedembryos, perhaps making detection of soluble ligands feasible. Forexample, embryos can be pre-fixed in 4% paraformaldehyde at 4° C.overnight, then treated with the AP-tagged protein in 1% Triton X-100.

Example 2 Epression Cloning of Elf-1 From Mouse Embryos

The strong reactivity of the presumptive midbrain and anterior hindbrainin the RAP-in situ of mouse embryos suggested that one or more ligandsfor mek4 and sek is expressed at high levels in this region. Wetherefore initiated an expression cloning strategy to isolate cDNA for aputative ligand expressed there. The appropriate region of the midbrainand hindbrain was excised from 80 mouse embryos at day 9.5 ofdevelopment. RNA was prepared and was used to construct a cDNA libraryin the eukaryotic expression vector CDM8 (Seed and Aruffo (1987) PNAS84:3365-3369). The library was produced as pools of approximately 1,000independent clones and was screened by a sib selection procedure. DNAfrom each pool was transiently transfected into a plate of COS cells.The cells were then tested by treating with mixed mek4-AP and sek-APsupernatants, washing, and staining for alkaline phosphatase activity insitu. After screening 36 pools, one positive pool was detected. Thispool was readily identifiable by the presence of intense AP stainingthat was coincident with the surfaces of several individual cellsscattered around the plate. The positive pool was subdivided andrescreened. and after a total of three rounds of screening, a singlepositive cDNA clone, Elf-1, was isolated.

Nucleotide sequencing of the positive cDNA clone revealed a single longopen reading frame that could encode a polypeptide of 209 amino acids(FIG. 2A. and SEQ ID Nos: 1 and 2). The open reading frame is followedby a typical 3′ untranslated sequence containing a polyadenylationsignal and a poly(A) tail. The N-terminus of the deduced proteinsequence begins with a methionine residue in a DNA sequence contextconsistent with a translation initiation site (Kozak, (1987) Nucl. AcidsRes 15:8125-8148), and is followed by an apparent signal sequence forpeptide secretion (von Heijne, (1990) J Membrane Biol 115:195-201). Thesecretion signal is followed by an amino acid sequence containing sixcysteine residues and three potential N-linked glycosylation sites. TheC-terminus ends with a stretch of fifteen predominantly hydrophobicamino acids (FIGS. 2A and 2B), suggesting the presence of a signal foraddition of a phosphatidylinositol glycan tail that could anchor thepolypeptide in the plasma membrane (Ferguson and Williams, (1 988) AnnuRev Biochem 57:285-320).

A search of the Genbank database did not reveal any sequences identicalor nearly identical to the polypeptide shown in FIG. 2A. Thispolypeptide appears to be a novel molecule which we have named Elf-1,for EPH ligand family-1. Two sequences in the database that did showobvious homology to Elf-1. One peptide, called B61, was identified in ascreen for mRNAs that could be induced by TNF-α treatment of endothelialcells (Holzman et al. (1990) Mol Cell Biol 10:5830-5838) and wasrecently shown to be a ligand for the Eck receptor tyrosine kinase(Bartley et al. (1994) Nature 368:558-561). Another ligand, LERK-2, hasbeen identified as a ligand for the elk receptor (Beckmann et al. (1994)EMBO J3:3757-3762). An alignment of the sequences of Elf-1 and B61demonstrates an amino acid identity of 45% overall. The sequenceconservation is strongest over the N-terminal 145 amino acids of thepredicted mature Elf-1 peptide, with a sequence identity of 53%. andconservation of all four cysteines in this part of the molecule. The GAPalignment of Elf-1 and LERK-2 gives an overall identity of 28%. TheC-terminal tail shows poor conservation of primary sequence amongts allthree proteins and in the Elf-1 protein, could serve primarily as alinker for attachment to the membrane.

The EPH family receptors themselves show a high degree of sequenceconservation. In particular the close sequence similarity of theirextracellular domains is unusual, generally showing identity in the40-60% range in pairwise alignments within the family. For comparison,the extracellular domains of the PDGF α and β receptors, which can bindthe same ligand, show 30% identity to one another, and 20% identity toc-kit, a member of the same subclass that binds a different ligand.Among the EPH family receptors, the Eck receptor is distant on thephylogenetic tree of the family from mek4 and sek, which are closelyrelated to one another (Maisonpierrc et al., (1993) Oncogene8:3277-3288, and Tuzi and Gullick (1994) Br, J Cancer 69:417-421). Thismakes it all the more noteworthy that Elf-1, LERK-2, and B61 aresomewhat related. The similarity of these ligands, particularly of theconserved cysteines, for relatively divergent members of the EPH familysuggests that other ligands for EPH family receptors may also prove tohave good conservation of primary sequence, but also that some overlapin receptor activation may be possible such that Elf-1 may have slightagonistic, and possibly even antagonistic activity when contacted withcells expressing EPH receptors more distantly related to the hek-relatedor sek-related receptors.

Example 3 Elf-1 is a Cell Surface Ligand for mek4-AP and sek-AP

A quantitative analysis of the binding of mek4-AP and sek-AP to cellsurface Elf-1 is shown in FIG. 3A-C. After transient transfection ofElf-1 into COS cells, binding of both mek4-AP and sek-AP to the cellsurface can be detected, indicating that both of these receptor fusionproteins can bind to Elf-1 on the cell surface (FIG. 3A). Whensaturating amounts of both mek4-AP and sek-AP are added simultaneously,the total AP binding is not additive, further confirming that they bindto the same ligand (FIG. 3A). A Scatchard analysis of the binding isshown in FIGS. 3B and 3C. For both mek4-AP and sek-AP the binding issaturable. Scatchard analyses produced values for the dissociationconstants of approximately 10⁻⁹ M for mek4-AP and approximately 10⁻⁸ Mfor sek-AP (FIGS. 3B and 3C).

A panel of cell lines was tested with mek4-AP and sek-AP for endogenousexpression of a cell surface ligand. Briefly, cells were treated withsupernatants containing mek4-AP, sek-AP or unfused AP as a control, eachat 1,000 OD/hr/ml. The cells were then washed. lysed and assayedcalorimetrically for bound AP activity. Some were found to be negativefor binding, while others gave binding that was well above the APcontrol background. The highest binding was shown by the rat liverstromal cell line BRL-3A, the mink lung fibroblast line Mv1Lu, and twoneural crest cell lines, NC7mycblue and NC7E1ablue. The BRL-3A cell linewas analyzed further. As in the case of COS cells transfected withElf-1, simultaneous treatment of BRL-3A cells with mek4-AP and sek-APdid not give additive binding of AP activity, indicating that the twofusion proteins bind to the same sites on the cell surface (FIG. 3D). AScatchard analysis of binding to BRL-3A cells (FIGS. 3E and 3F)indicated binding affinities for both mek4-AP and sek-AP that aresimilar to those found with Elf-1 in COS cells, consistent with Elf-1being a ligand expressed on BRL-3A cells. The number of binding sitesper BRL-3A cell is approximately 50,000. In situ staining was also usedto examine the cell surface binding of mek4-AP and sek-AP to BRL-3Acells, and indicated expression of the ligand over the whole cellsurface. It is interesting to note that RAP-in situ staining of themouse embryo midbrain/hindbrain region was obvious within two minutes,whereas comparable staining of the BRL-3A cells took several hours todevelop. This difference of approximately two orders of magnitudesuggests that the ligand polypeptide is present at remarkably highlevels in the embryo.

The binding affinities measured here were determined for the interactionbetween a tagged soluble receptor and a membrane bound ligand, ratherthan the more usual measurement between a membrane bound receptor and asoluble ligand labeled by chemical modification. However, otherAP-tagged receptors or ligands have produced measured affinities in linewith expected values (for example, Flanagan and Leder, supra; Morrisonand Leder, (1992) J Biol Chem 267:11957-11963; and Ornitz et al.,supra). Moreover, in the case of a receptor and a ligand which are bothmembrane bound in their native state neither type of measurement maytruly reflect the avidity of the interaction in vivo, though both typesof measurement can give some indication of the likely strength ofthisinteraction.

The K_(D) of approximately 10⁻⁹ M for the binding of Elf-1 to mek4-AP iswithin the typical range of affinities for ligands binding to theircognate receptor tyrosine kinases making it likely that this representsa genuine, biologically significant ligand-receptor interaction. TheK_(D) estimate of 10⁻⁸ M for Elf-1 binding to sek-AP is an affinitylower than many known receptor-ligand interactions, though not all. Inthis context it is worth noting that a similar, or slightly weaker,K_(D) of approximately 2-3×10⁻⁸ M was reported for the interaction ofthe Eck receptor with its kinase-activating ligand B61 (Baitley et al.,supra).

Several additional factors are relevant in considering the potentialbiological significancc of such an interaction. First, the avidity ofthe interaction between a receptor and a ligand that are both presentedat the cell surface is likely to be greatly enhanced by the cooperativeeffect of highly multivalent ligand-receptor binding between two apposedmembranes. Second, our results suggest that Elf-1 is present at veryhigh localized concentrations in the embryo, another factor that wouldfavor interaction with a receptor of moderate affinity. Additionalsupport for the biological significance of the interaction of Elf-1 withmek4 and sek comes from our in sitit studies of mouse embryos. RAP-insitu with either mek4-AP or sek-AP detected a pattern of liganddistribution that was subsequently found to be strikingly similar to thepattern of Elf-1 RNA expression. Furthermore, the in situ RNAhybridization results, described further below, indicated that inseveral regions of the midgestation embryo, mek4 and/or sek areexpressed in patterns that are complementary to the areas of Elf-1expression, providing further evidence in support of interactionsbetween these molecules during development. It is also possible thatElf-1 may bind to other EPH family receptors or that mek4 and sek mayhave additional ligands.

Example 4 Elf-1 Attachment to the Cell Surface is Sensitive to PI-PLC inCell Lines and Embryos

The hydrophobic C-terminus of Elf-1 suggested a signal for covalentlinkage to a phosphatidylinositol glycani moiety. Such a linkage couldaccount for the observed association of Elf-1 with cell surfaces. Thispossibility was tested by treatment with phosphatidylinositol-specificphospholipase C (PI-PLC), an enzyme that cleaves phosphatidylinositollinkages and might therefore be expected to release Elf-1 from the cellsurface. mek4-AP and sek-AP binding activity is removed from the cellsurface after PI-PLC treatment of either COS cells transfected withElf-1, or BRL-3A cells. Bindinig activity of the ligand is not destroyedby this treatment, as co-immunioprecipitation experiments indicated thatPI-PLC treatment also results in release of ligand polypeptide into thecell supernatant. It therefore appears that cell surface Elf-1 intransfected COS cells, and the cell surface ligand expressedendogenously in BRL-3A cells, are attached to the cell membrane via aphosphatidylinositol glycan linkage.

The effect of PI-PLC was also tested on embryos. In RAP-in situexperiments, prior treatment of the embryos with PI-PLC resulted in areduction of mek4-AP or sek-AP staining to background levels. In theembryo too, it therefore appears that the ligand(s) detected by mek4-APand sek-AP are held in place by an association with cell surfaces via aphosphatidylinositol glycan linkage.

Example 5 In situ Hybridization Analysis of Elf-1 RNA Expression inMouse Embryos

The RAP-in situ experiments with mek4-AP and sek-AP indicated that thesereceptors can bind to a ligand in mouse embryos. It was therefore ofinterest to compare the RAP-in situ results with the expression patternof Elf-1 determined by RNA hybridization in situ. If Elf-1 is indeed aligand detected by the RAP-in situ procedure it would be expected thatthe RNA hybridization in situ and the RAP-in situ should overlap in atleast some areas.

An RNA probe was prepared from the 3′ end of the Elf-1 clone and wasused to examine mouse embryos by whole mount in situ hybridization. Theexpression pattern detected by the Elf-1 RNA probe in day 9.5 embryoswas similar to that seen in the RAP-in situ experiments with mek4-AP andsek-AP. The strongest expression was again seen in the region of thepresumptive midbrain and anterior hindbrain. As in the RAP-in situexperiments, the color development in this region was rapid, beingreadily visible in less than ten minutes, consistent with a high levelof Elf-1 RNA expression. Other areas of RNA expression were also verysimilar to those seen in the RAP in situ, although no obvious signal wasseen with the Elf-1 RNA hybridization probe over the dorsal face of thepresumptive spinal cord, where a prominent stripe of reactivity was seenwith the mek4-AP and sek-AP reagents.

To obtain further information on the potential roles of Elf-1, mek4 andsek in the embryo, the expression patterns of all three molecules werecompared by whole mount in situ hybridization of embryos at days 8.5,9.5, and 10.5 of development. We present our initial observations here.The sek expression pattern has also been described more extensivelyelsewhere (Nieto et al. (1992) Development 116:11137-1150). In severalseparate developmental fields of the embryo, the ligand and the tworeceptors are expressed in patterns that are adjacent and complementary,implying roles for their interactions in patterning of these areas.

These regions include the neural tube, branchial arches, somites andlimb buds. Expression of Elf-1 and sek RNA is seen by the earliest timepoint analyzed (day 8.5). Expression of mek4 RNA was less obvious atthis time point, but was present at high levels by day 9.5. In theneural tube, a region of very high Elf-1 expression in the midbrain andanterior hindbrain (metencephalon) was flanked on one side by a regionof high mek4 and sek expression near the forebrain-midbrain junction,and on the other side by regions of mek4 and sek expression in thehinidbrain. Expression of sek in this region is especially high inrhombomeres 3 and 5, as reported previously (Nieto et al., supra). Theexpression of mek4 also appears to be highest in different rhombomeres.Elf-1 RNA expression in the somites, branchial arehes and limb budsappears in a broad, diffuse pattern at this level of analysis. The sekand mek4 receptors are expressed in these same structures, and withineach are restricted to smaller subregions. Thus, in the somites, mek4 isrestricted to the dorsal and/or ventral parts, while sek is expressed ineach new condensing somite in the day 8.5 embryo, and also in the dorsalpart of each somite at later stages. In the limb buds, sek is expressedin a band at the distal tip while mek4 is expressed in a posteriorproximal region, as well as in the lateral plate between the limb buds.The expression in the branchial arches is complex, but again mek4 andsek appear in localized subregions.

Example 6 Detection of Genomic Elf-1 Genes in Both Mouse and Human Cells

We have performed a genomic Southern blot on both mouse and human cDNA.Six samples of genomic DNA from each species were cut with six differentrestriction enzymes (Xhol, BamHI, ScaI, XbaI, EcoRI, and NotI). Theprobe was a 1.1 kb fragment of the Elf-1 clone extending from a PstIsite in the middle of the Elf-1 coding sequence to the 3′ end of thecDNA. The blots were washed at high stringency (0.1×SSC, 65° C.), and ineach sample for both species. a single prominant hybridizing band wasdetected.

Experimental Procedures

A. Construction and Expression of AP Fusion Proteins

To produce the mek4-AP and sek-AP fusion constructs, sequences encodingthe extracellular domains were amplified by polymerase chain reaction.For mek4-AP the sequence from nucleotide 32 to 1708 (Sajjadi et al.,(1991) New Biol 3:769-778; Genbank accession M685113) was amplified frommouse brain cDNA, and for sek-AP the sequence from nucleotide 12 to 1698(Gilardi-Hebenstrcit et al., (1992) Oncogene 7:2499-2506; Genbankaccession X65138) was amplified from cDNA of NIH-3T3 cells. Restrictionsites at the ends of the amplification primers were cleaved with BamHIand inserted into the BglII site of the vector APtag-1 (Flanagan andLeder, supra) so that each receptor extracellular domain was fused tosecreted human placental alkaline phosphatase through a four amino acidlinker (Gly-Ser-Ser-Gly).

Each plasmid was linearized with PvuI, and 2 μg (more can give lowerexpression) was stably tranisfected with 0.5 μg of pSV7neo selectionplasmid and 20 μg of calf thymus carrier DNA by calcium phosphateprecipitation into a 10 cm dish of NIH-3T3 cells. Cells were cloned in96-well plates during G418 selection and high-expressers were selectedby testing supernatants for AP activity in a 96-well plate reader asdescribed (Flanagan and Leder, supra) except that L-homoarginine wasomitted from all AP assays here. Clones expressing approx. 1,000 OD/hrof AP activity per ml of supernatant were used as a source of mek4-APand sek-AP proteins. Cells were grown to confluence, then mediaconditioned for a further 3 days were centrifuged, 0.45 μm filtered andstored at 4° C. with 20 mM HEPES pH7.0 and 0.05% sodium azidc. APactivities are expressed here as OD units per hour (OD/hr ), indicatingthe rate of hydrolysis of the chromogenic substrate p-nitropheniylphosphate. One picomole of AP fusion protein corresponds to an activityof approximately 30 OD/hr under the conditions of the assay.

B. RAP In Situ of Mouse Embryos

Embryos from Swiss-Webster mice were transferred to 1.5 ml microfugetubes and rinsed once in HBHA buffer (Hank's balanced salt solution with0.5 mg/ml BSA, 0.1% NaN₃, 20 mM HEPES pH7.0). The embryos were thenincubated in tissue culture supernatants containing RAP fusion proteins,or AP as a control, for 75 min on a rotator at room temperature. Theywere then washed six times in HBHA buffer, treated for 2.5 min with anacetone/formaldehyde fixative (60% acetone, 3% formaldehyde, 20 mM HEPESpH 7.0), washed three times in HBS (150 mM NaCl, 20 mM HEPES pH7.0), andthen incubated in a 65° C. water bath for 15 min, inactivatingendogenous cellular phosphatases but not the uniquely heat stable AP ofthe fusion proteins. After rinsing with AP buffer (100 mM Tris-HCl pH9.5, 100 mM NaCl, 5 mM MgCl₂), the cells were stained for 5-10 min inthe same buffer containing 0.17 mg/ml BCIP and 0.33 mg/ml NBT.

C. Expression Library Construction and Screening

Eighty embryos at day 9.5 of development were obtained from sixSwiss-Webster mice. Embryos were rinsed in PBS and the region of themidbrain and anterior hindbrain previously found to stain strongly withmek4-AP and sek-AP by RAP in situ was cut from each embryo under adissecting microscope. RNA was prepared by the single-step method(Chomczynski et al. (1987) Anal. Biochem 162:156-159) and after oneround of oligo(dT) cellulose purification the yield was 4 μg. Doublestranded cDNA was prepared (Invitrogen kit), inserted into theexpression vector CDM8 (Seed et al. (1987) PNAS 84:3365-3369) andtransfected into E. coli. Pools of approximately 1,000 clones wereplated on nitrocellulose filters and then replica plated. DNA wasprepared from one replica and the other was stored.

To screen the library, DNA of each pool was transiently transfected intoa 10 cm dish of COS cells using Lipofectamine as described by themanufacturer (Gibco-BRL). After 48 hr the cells, just at or beforereaching confluence, were washed once in HBHA buffer and then incubatedin an equal mixture of sek-AP and sek-AP supernatants for 90 min at roomtemperature. Plates were then washed six times in HBHA buffer, treatedfor 30 sec with acetone/formaldehyde fixative, and washed twice in HBS.Uniform heating to inactivate endogenous cellular phosphatases iscritical and was achieved by incubating plates containing 10 ml HBS in asingle layer on a flat shelf in a 65° C. oven for 100 min. Plates werethen rinsed with AP buffer and stained for AP activity for 0.5-12 hr inthe same buffer containing 0.17 mg/ml BCIP and 0.33 mg/ml NBT. Stainingwas monitored periodically against a white background under a dissectingmicroscope.

After identificalon of a positive pool, the stored library filter wasreplica plated again, and rescreening was performed with colonies fromsuccessively smaller areas of the filter. After a total of three roundsof screening, DNA was prepared from single colonies and a positive clonewas identified.

D. Qualntitaive Cell Surfce Binding Assays, Cell Culture, and PI-PLCTreatment

Quantitative cell surface binding assays with mek4-AP and sek-AP wereperformed essentially as described previously for Kit-AP (Flanagan andLeder, supra; Flanagan et al. supra). Cells in 10 cm plates were washedwith HBHA buffer and then incubated at room temperature withsupernatants containing mek4-AP or sek-AP fusion proteins or AP as acontrol. For Scatchard binding analyses the supernatants wereconcentrated in an ultrafiltration cell (Amicon) and dilutions were madewith HBHA buffer. After 90 min, the cells were washed six times withHBHA buffer, lysed, and assayed colorimetrically for bound alkalinephosphatase activity.

Cells were grown in DMEM with 10% bovine calf serum. Cell lines areavailable from the American Type Culture Collection, except the NC7lines, established by oncogene-mediated immortalization of explantedmouse neural crest and BMS-12, a stromal line established by passagingof mouse bone marrow.

For experiments to assess phosphatidylinositol linkage. cells or embryoswere pretreated in complete tissue culture medium at 37° C. for 2 hrwith 100-300 mU/ml of Pl-PLC (Sigma).

E. Co-immunoprecipitation with Receptor-AP Fusion Proteins

Ligand polypeptides were analyzed by co-immunoprecipitation withmek4-AP. Cells in 6-well plates were metabolically labeled with³⁵S-methionine as described (Flanagan et al., supar). The supernatants.concentrated to 200 μl on a Centricon-10 (Amicon), and cells, lysed in200 μl of lysis buffer (1% Triton-X100, 10 mM Tris-HCl pH 7, 1 mM PMSF)were then incubated for 90 min at room temperature with an equal volumeof supernatant containing mek4-AP or AP. Labeled li,and polypeptideswere then co-immunoprecipitated with the mek4-AP fusion protein usinig amonoclonal antibody against human placental alkaline phosphatase (Cat.no. MIA 1801, Medix Biotech, Foster City, Calif.) as described (Flanaganet al., supra).

F. In situ RATA Hybridization of Mouse Embryos

Whole mount in situ hybridization of mouse embryos was pertormcd asdescribed (In situ hvbridization: a practical approach, D. G. Wilkinson,ed. (Oxford University Press), pp. 75-83) except that post-hybridizationwashes were done three times in solution 1 and three times in solution3, without using solution 2 or ribonuclease. RNA probes were prepared bytranscription from promoters in the Bluescript (Stratagene) or CDM8(Seed et al., supra) plasmids. The Elf-1 anisense probe was a 1 kbfragment extending from a unique Pst1 site to the 3′ end of Elf-1. Thesek antisense probe was a 0.8 kb fragment from a BsmI site at nucleotide878 to the transmembrane sequence at nucleotide 1698, and the senseprobe was a 0.8 kb fragment from a HindIII site at nucleotide 879 tonucleotide 12 at the 5′ end (Gilardi-Hebenstreit et al. (1992) Oncogene7:2499-2506; Genbank accession X65138). The mek4 antisense probe was a1.1 kb fragment from a BsmI site at nucleotide 564 to the transmembranesequence at nucleotide 1708, and the sense probe was a 0.8 kb fragmentfrom a HindIII site at nucleotide 897 to nucleotide 32 at the 5′ end(Sajjadi et al. (1991) New Biol 3:769-778; Genbank accession M68513).

All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

2 1615 base pairs nucleic acid both linear cDNA not provided CDS 10..636sig_peptide 10..69 1 ACCGGGGCC ATG GCG CCC GCG CAG CGC CCG CTG CTG CCGCTG CTG CTG 48 Met Ala Pro Ala Gln Arg Pro Leu Leu Pro Leu Leu Leu 1 510 CTG CTG CTG CCG CTG CGT GCG CGC AAC GAG GAC CCG GCC CGG GCC AAC 96Leu Leu Leu Pro Leu Arg Ala Arg Asn Glu Asp Pro Ala Arg Ala Asn 15 20 25GCT GAC CGA TAC GCA GTC TAC TGG AAC CGT AGC AAC CCC AGG TTT CAG 144 AlaAsp Arg Tyr Ala Val Tyr Trp Asn Arg Ser Asn Pro Arg Phe Gln 30 35 40 45GTG AGC GCT GTG GGT GAT GGC GGC GGC TAT ACC GTG GAG GTG AGC ATC 192 ValSer Ala Val Gly Asp Gly Gly Gly Tyr Thr Val Glu Val Ser Ile 50 55 60 AACGAC TAC CTG GAT ATC TAC TGC CCA CAC TAC GGG GCG CCG CTG CCC 240 Asn AspTyr Leu Asp Ile Tyr Cys Pro His Tyr Gly Ala Pro Leu Pro 65 70 75 CCG GCTGAG CGC ATG GAG CGG TAC ATC CTG TAC ATG GTG AAT GGT GAG 288 Pro Ala GluArg Met Glu Arg Tyr Ile Leu Tyr Met Val Asn Gly Glu 80 85 90 GGC CAC GCCTCC TGT GAC CAC CGG CAG CGA GGC TTC AAG CGC TGG GAA 336 Gly His Ala SerCys Asp His Arg Gln Arg Gly Phe Lys Arg Trp Glu 95 100 105 TGC AAC CGGCCC GCA GCG CCC GGG GGA CCC CTC AAG TTC TCA GAG AAG 384 Cys Asn Arg ProAla Ala Pro Gly Gly Pro Leu Lys Phe Ser Glu Lys 110 115 120 125 TTC CAACTC TTC ACC CCC TTT TCC CTG GGC TTT GAG TTC CGG CCT GGC 432 Phe Gln LeuPhe Thr Pro Phe Ser Leu Gly Phe Glu Phe Arg Pro Gly 130 135 140 CAC GAATAC TAC TAC ATC TCT GCC ACA CCT CCC AAC CTC GTG GAC CGA 480 His Glu TyrTyr Tyr Ile Ser Ala Thr Pro Pro Asn Leu Val Asp Arg 145 150 155 CCC TGCCTG CGA CTC AAG GTT TAT GTG CGT CCA ACC AAT GAG ACC CTG 528 Pro Cys LeuArg Leu Lys Val Tyr Val Arg Pro Thr Asn Glu Thr Leu 160 165 170 TAT GAGGCT CCA GAG CCC ATC TTC ACC AGT AAC AGC TCC TGC AGC GGC 576 Tyr Glu AlaPro Glu Pro Ile Phe Thr Ser Asn Ser Ser Cys Ser Gly 175 180 185 CTG GGTGGC TGC CAC CTC TTC CTC ACC ACC GTC CCT GTG CTG TGG TCC 624 Leu Gly GlyCys His Leu Phe Leu Thr Thr Val Pro Val Leu Trp Ser 190 195 200 205 CTTCTG GGC TCC TAGTGTCAGG CCGGAGAACA CCAGCCCCAC CTGGACCCCG 676 Leu Leu GlySer TGACCTTTGC CCTCTGACCT GCCACGGCCA CCTCCGAGAC AAAATCCTTG CTGCTTCTCT736 TTCATGGTGC TGTCCCGCCG GAGGAGGCCA TCCATCCGTC CCTGGGATGC AACATGGGGT796 CCCAATGCCT GAGGAGAAGA CCCCCCCCCA AGGCTGACTC GCTTTCACCA GGCCACCAGG856 GCCATCCAGT GTTGTTTAAT TACAGTCGGA AAGACTTAAG GTTTTTCTTT TAATTTAATT916 TATTCCCTGA CATTGCTGGT GACACTGGGA AGGGAACAAG CCACAGGGAT GAGGTGAAGC976 CATCTCTGTC CTTCCTGGAA TACCGGAGAT CCAGGGGCCT CCAGCTGCTC CTTTCTTCTG1036 TGTCCTGTTA TTTGGGTCCC AGATGGAGCC CACCGCGGAC TTGCCTTGCA TTCCTCAGGC1096 CAGGCAAGCC TGAGCCAGAA AGGGGGCACG GTGCCAGCCT CTCGGGGACT CTGGGGGTGC1156 CATCCCCCAC TCTTCTTCCA GCCACTCTCG GGCCCCACTC CCACATCATC TCAGAAACCC1216 TTCAGCCCTC GCAACTCGCC CCTCCGGGCC CCCCCACCAG GCACAACCAT CCCCGGGGCC1276 AGCCGGGACG TTGTCGGTTT ATTTCTGTAA ATAGAAACCA GCAAGTGTAT ACTGTGATTT1336 ATTTTAATGT ATTCTTAAGG ACAGAATGGA AATTCTTTAA AAAAAATTTT TTTTCCGACC1396 TTCAATTCAA GGGGTCATTT ATTTTGGTGG GGGGAGTGGG GTGGACTTTT TAGGATAGAA1456 GCAACACTTT GCAATAAACT CATTTTTTTT TGTTCCGTTG GAGCCCTCCC CCTTGATCAT1516 GTGACCTAGT AATGTTTATA ACAAAAAAAA AAAAAAAAGA GTAAACCAGT CCTAACCAGA1576 TTCAAAATTT CGTTACAGAA AGTATTCATA TTCTATTCT 1615 209 amino acidsamino acid linear protein not provided 2 Met Ala Pro Ala Gln Arg Pro LeuLeu Pro Leu Leu Leu Leu Leu Leu 1 5 10 15 Pro Leu Arg Ala Arg Asn GluAsp Pro Ala Arg Ala Asn Ala Asp Arg 20 25 30 Tyr Ala Val Tyr Trp Asn ArgSer Asn Pro Arg Phe Gln Val Ser Ala 35 40 45 Val Gly Asp Gly Gly Gly TyrThr Val Glu Val Ser Ile Asn Asp Tyr 50 55 60 Leu Asp Ile Tyr Cys Pro HisTyr Gly Ala Pro Leu Pro Pro Ala Glu 65 70 75 80 Arg Met Glu Arg Tyr IleLeu Tyr Met Val Asn Gly Glu Gly His Ala 85 90 95 Ser Cys Asp His Arg GlnArg Gly Phe Lys Arg Trp Glu Cys Asn Arg 100 105 110 Pro Ala Ala Pro GlyGly Pro Leu Lys Phe Ser Glu Lys Phe Gln Leu 115 120 125 Phe Thr Pro PheSer Leu Gly Phe Glu Phe Arg Pro Gly His Glu Tyr 130 135 140 Tyr Tyr IleSer Ala Thr Pro Pro Asn Leu Val Asp Arg Pro Cys Leu 145 150 155 160 ArgLeu Lys Val Tyr Val Arg Pro Thr Asn Glu Thr Leu Tyr Glu Ala 165 170 175Pro Glu Pro Ile Phe Thr Ser Asn Ser Ser Cys Ser Gly Leu Gly Gly 180 185190 Cys His Leu Phe Leu Thr Thr Val Pro Val Leu Trp Ser Leu Leu Gly 195200 205 Ser

We claim:
 1. An isolated or recombinantly produced naturally occurringElf-1 polypeptide encoded by a nucleic acid which hybridizes understringent conditions to a nucleic acid having the sequence representedin SEQ ID No. 1, and which polypeptide specifically binds to an EPH-typereceptor.
 2. An isolated or recombinantly produced naturally occurringElf-1 polypeptide comprising the amino acid sequence represented in SEQID No. 2 which polypeptide specifically binds to an EPH-type receptor.3. The Elf-1 polypeptide of claim 1 or 2, wherein said polypeptidemodulates at least one of proliferation, differentiation, and survivalof a cell which expresses said EPH-type receptor.
 4. The Elf-1polypeptide of claim 1 or 2, wherein said polypeptide modulatesintracellular signal transduction pathways mediated by said EPH-typereceptor.
 5. An isolated or recombinantly produced Elf-1 polypeptidecomprising an amino acid sequence corresponding to Cys-69 throughCys-159 of the amino acid sequence represented by SEQ ID No.
 2. 6. Anisolated or recombinantly produced Elf-1 polypeptide comprising at leastan amino acid sequence corresponding to Arg-21 through Thr-182 of theamino acid sequence represented by SEQ ID No.
 2. 7. An isolated orrecombinantly produced Elf-1 polypeptide which differs from a naturallyoccurring Elf-1 polypeptide only in being post-translationally modifiedto include a covalently added moiety selected from the group consistingof a phosphatidylinositol and a carbohydrate, wherein said naturallyoccurring Elf-1 polypeptide is encoded by a nucleic acid whichhybridizes under stringent conditions to a nucleic acid having thesequence represented by SEQ ID NO.
 1. 8. The Elf-1 polypeptide of claims1 or 2, wherein said polypeptide is a fusion protein further comprisinga second polypeptide sequence having an amino acid sequence unrelated tothe amino acid sequence represented by SEQ ID NO.
 2. 9. The Elf-1polypeptide of claim 8, wherein said fusion protein includes a secondpolypeptide sequence selected from the group consisting of a polypeptidewhich has an enzymatic activity, a polypeptide which functions as adetectable label for detecting the presence of said fusion protein, anda polypeptide which functions as a matrix-binding domain forimmobilizing said fusion protein.
 10. The Elf-1 polypeptide of claim 1or 2, wherein the EPH-type receptor is a HEK receptor.
 11. The Elf-1polypeptide of claim 10, wherein said EPH-type receptor is selected fromthe group consisting of mek4-related receptors and sek-relatedreceptors.